EN 2000-32-EC

I

(Actswhosepublicationisobligatory)

COMMISSIONDIRECTIVE2000/32/EC

of19May2000

adaptingtotechnicalprogressforthe26thtimeCouncilDirective67/548/EEContheapproximationofthelaws,regulationsandadministrativeprovisionsrelatingtotheclassification,

packagingandlabellingofdangeroussubstances(*)

(TextwithEEArelevance)

THECOMMISSIONOFTHEEUROPEANCOMMUNITIES,

(4)AnnexIXtoDirective67/548/EECcontainstheprovisions

HavingregardtotheTreatyestablishingtheEuropeanCommunity,

HavingregardtoCouncilDirective67/548/EECof27June1967ontheapproximationofthelaws,regulationsandadministrativeprovisionsrelatingtotheclassification,packagingandlabellingofdangeroussubstances(1),aslastamendedbyEuropeanParliamentandCouncilDirective1999/33/EC(2),andinparticularArticle28thereof,Whereas:

(1)AnnexItoDirective67/548/EECcontainsalistof

relatingtochild-prooffastenings.Thoseprovisionsshouldbeadaptedandupdated.Itisnecessarytoextendthescopeoftheuseofchild-prooffastenings.

(5)ThemeasuresprovidedforinthisDirectivearein

accordancewiththeopinionoftheCommitteeontheAdaptationtoTechnicalProgressoftheDirectivesfortheEliminationofTechnicalBarrierstoTradeinDangerousSubstancesandPreparations,

HASADOPTEDTHISDIRECTIVE:

dangeroussubstances,togetherwithparticularsoftheclassificationandlabellingofeachsubstance.PresentscientificandtechnicalknowledgehasshownthatthelistofdangeroussubstancesinthatAnnexshouldbeadapted.CertainlanguageversionsoftheDirectiverequirecorrectionsinspecificsectionsoftheforewordandofTableAtoAnnexI.

Article1

Directive67/548/EECisherebyamendedasfollows:1.AnnexIisamendedasfollows:

(a)NoteQinAnnex1AtothisDirectivereplacesthe

correspondingnoteintheForeword.(b)TherowsinAnnex1BtothisDirectivereplacethe

correspondingrowsinTableA.(c)TheentriesinAnnex1CtothisDirectivereplacethe

correspondingentries.(d)TheentriesinAnnex1DtothisDirectiveareinserted.2.TheriskphraseinAnnex2tothisDirectivereplacesthe

correspondingphraseinAnnexIII.3.AnnexIVisamendedasfollows:

(a)ThesafetyphrasesinAnnex3AtothisDirective

replacethecorrespondingphrasesinAnnexIV.

(2)AnnexIIItoDirective67/548/EECcontainsalistof

phrasesindicatingthenatureofspecialrisksattributedtodangeroussubstancesandpreparations.AnnexIVtoDirective67/548/EECcontainsalistofthephrasesindicatingthesafetyadviceconcerningdangeroussubstancesandpreparations.AnnexVItoDirective67/548/EECcontainsaguidetotheclassificationandlabellingofdangeroussubstancesandpreparations.CertainlanguageversionsoftheDirectiverequirecorrectionsinspecificsectionsofAnnexesIII,IVandVI.

(3)AnnexVtoDirective67/548/EEClaysdownthemethods

forthedeterminationofthephysico-chemicalproperties,toxicityandecotoxicityofsubstancesandpreparations.ItisnecessarytoadaptthatAnnextotechnicalprogress.

(*)Adoptedafterthe27thadaptation.(1)OJ196,16.8.1967,p.1.(2)OJL199,30.7.1999,p.57.

L136/2ENOfficialJournaloftheEuropeanCommunities

Article2

8.6.2000

(b)ThecombinedsafetyphrasesinAnnex3Btothis

DirectivereplacethecorrespondingphrasesinAnnexIV.4.PartBofAnnexVisamendedasfollows:

(a)ThetextinAnnex4AtothisDirectivereplaces

ChapterB.10.(b)ThetextinAnnex4BtothisDirectivereplaces

ChapterB.11.(c)ThetextinAnnex4CtothisDirectivereplaces

ChapterB.12.(d)ThetextinAnnex4DtothisDirectivereplaces

ChaptersB.13andB.14.(e)ThetextinAnnex4EtothisDirectivereplacesChapter

B.17.(f)ThetextinAnnex4FtothisDirectivereplacesChapter

B.23.ThetitleofChapterB.23intheexplanatorynoteischangedaccordingly.(g)ThetextinAnnex4GtothisDirectiveisadded.5.ThefourthindentofthegeneralintroductiontoPartCof

AnnexVisdeleted.6.ThetextsinAnnex5tothisDirectivereplacethe

correspondingtextsinAnnexVI.7.AnnexIXisamendedassetoutinAnnex6tothis

Directive.

1.MemberStatesshallbringintoforcethelaws,regulationsandadministrativeprovisionsnecessarytocomplywiththisDirectiveby1June2001atthelatest.TheyshallforthwithinformtheCommissionthereof.

WhenMemberStatesadoptthoseprovisions,theyshallcontainareferencetothisDirectiveorbeaccompaniedbysuchareferenceontheoccasionoftheirofficialpublication.MemberStatesshalldeterminehowsuchreferenceistobemade.

2.MemberStatesshallcommunicatetotheCommissionthemainprovisionsofnationallawwhichtheyadoptinthefieldcoveredbythisDirectiveandacorrelationtablebetweenthisDirectiveandthenationalprovisionsadopted.

Article3

ThisDirectiveshallenterintoforceonthethirddayfollowingitspublicationintheOfficialJournaloftheEuropeanCommunities.

Article4

ThisDirectiveisaddressedtotheMemberStates.DoneatBrussels,19May2000.

FortheCommission

MargotWALLSTRÖM

MemberoftheCommission

8.6.2000ENOfficialJournaloftheEuropeanCommunitiesL136/3

ANNEX1A

FOREWORDTOANNEXI

Explanationofthenotesrelatingtotheidentification,classificationandlabellingofsubstances

DA:NoteQ:

Klassificeringensomkræftfremkaldendekanudeladesforfibre,somopfylderenaffølgendebetingelser:

󰂗enkortvarigbiopersistensprøvevedinhalationharvist,atfibre,dererlængereend20µm,harenvægtethalve-ringstidpåmindreend10dage󰂗enkortvarigbiopersistensprøvevedintratrakealinstillationharvist,atfibre,dererlængereend20µm,haren

vægtethalveringstidpåmindreend40dage󰂗enegnetintra-peritonealprøveikkeharvistkræftfremkaldendevirkning,eller

󰂗enegnetlangvariginhalationsprøveikkeharvistrelevantesygdomsfremkaldendevirkningerellerneoplastiske

forandringer.

SV:NoteQ:

Ämnetbehöverinteklassificerassomcancerframkallandeomdetkanvisasattdetuppfyllerettavföljandevillkor:󰂗ettkorttidstestförattbestämmadenbiologiskabeständighetenvidinhalationharvisatattfibrerlängreän20µm

harenviktadhalveringstidpåmindreän10dagar󰂗ettkorttidstestförattbestämmadenbiologiskabeständighetenvidintratrakealinstillationharvisatattfibrerlängre

än20µmharenviktadhalveringstidpåmindreän40dagar󰂗ettlämpligtintraperitonealttestharintegivitbeläggförförhöjdcancerogenitet

󰂗frånvaroavrelevantpatogenitetellerneoplastiskaförändringariettlämpligtlångtidsinhalationstest.(DoesnotconcerntheESversion)(DoesnotconcerntheDEversion)(DoesnotconcerntheELversion)(DoesnotconcerntheENversion)(DoesnotconcerntheFRversion)(DoesnotconcerntheITversion)(DoesnotconcerntheNLversion)(DoesnotconcernthePTversion)(DoesnotconcerntheFIversion)

ANNEX1B

TABLEA

Z

Symb.

ES

DA

DE

EL

EN

FI

FR

IT

NL

PT

SV

󰂑18ArArgónArgonArgonAqcüArgonArgonArgonArgonArgonÁrgonArgon󰂒󰂑

Gd

Gadolinio

Gadolinium

Gadolinium

Cadokßmio

Gadolinium

Gadolinium

Gadolinium

Gadolinio

Gadolinium

Gadolínio

Gadolinium󰂒

L136/4ENOfficialJournaloftheEuropeanCommunities8.6.2000ANNEX1C

IndexNoChemicalnamerelatedNotestoECNoCASNoClassificationLabellingConcentrationlimits

Notessubstances

preparations

relatedto006-011-00-7

carbaryl1-naphthyl(ISO)

methylcarbamate200-555-063-25-2

Carc.Xn;Cat.3;Xn;N;R50R22R40R:N

S:(2-)22-24-36/37-46-6122-40-50

006-013-00-8metam-sodiumsodiummethyldithiocarbamate

(ISO)

205-293-0137-42-8

Xn;R31R22C;C;R:N

R43

R34S:(1/2-)26-36/37/39-45-60-61

22-31-34-43-50/53

N;R50-53006-015-00-9

diuron3-(3,4-dichlorophenyl)-1,1-dimethylurea

(ISO)

206-354-4330-54-1

Carc.Muta.Cat.3;R40Xn;Xn;Cat.3;R40R:N

N;R50-53R22-48/22S:(2-)13-22-23-37-46-60-6122-40-48/22-50/53

006-016-00-4

propoxur2-isopropoxyphenyl(ISO)

204-043-8114-26-1

T;N;R25R50-53T;2-isopropoxyphenylNmethylcarbamate-methylcarbamateR:N

S:(1/2-)37-45-60-6125-50/53

006-017-00-Xaldicarb2-methyl-2-(methylthio)propanal-(ISO)

O-204-123-2116-06-3

T+;T;R26/28T+;(N-methylcarbamoyl)oxime

N;R24R50-53R:N

S:(1/2-)22-36/37-45-60-6124-26/28-50/53

006-018-00-5aminocarb4-dimethylamino-3-tolyl(ISO)

methylcarbamate217-990-72032-59-9

T;N;R24/25R50-53T;R:N

S:(1/2-)28-36/37-45-60-6124/25-50/53

006-019-00-0di-allateS218-961-12303-16-4

Carc.Xn;Cat.3;R40Xn;diisopropylthiocarbamate

-(2,3-dichloroallyl)-(ISO)

N,N-N;R50-53R22R:S:(2-)25-36/37-60-6122-40-50/53

N

006-020-00-6barban4-chlorbut-2-ynyl(ISO)

N-(3-chlorphenyl)carbamate202-930-4101-27-9

Xn;R43

R22Xn;N;R50-53R:N

S:(2-)24-36/37-60-6122-43-50/53

006-023-00-2mercaptodimethurmethiocarb

(ISO)217-991-22032-65-7

T;N;R25R50-53

T;3,5-dimethyl-4-methylthiophenylR:N

N-methylcarbamate

S:(1/2-)22-37-45-60-6125-50/53

006-024-00-8proxan-sodiumsodiumO-isopropyldithiocarbamate

(ISO)

205-443-5140-93-2

Xn;Xi;R22Xn;N;R51-53

R38R:S:(2-)13-61

22-38-51/53N

8.6.2000ENOfficialJournaloftheEuropeanCommunitiesL136/5IndexNoChemicalname

substances

relatedNotestoECNoCASNoClassificationLabellingConcentrationlimits

Notespreparations

relatedto006-026-00-9

carbofuran2,3-dihydro-2,2-dimethylbenzofuran-7-yl(ISO)

216-353-01563-66-2

T+;N-methylcarbamate

N;R50-53

R26/28T+;R:S:(1/2-)36/37-45-60-61

26/28-50/53

N

006-028-00-Xdinobuton2-(1-methylpropyl)-4,6-dinitrophenyl(ISO)

213-546-1973-21-7

T;carbonate

isopropylN;R25R50-53

T;R:N

S:(1/2-)37-45-60-6125-50/53

006-029-00-5dioxacarb2-(1,3-dioxolan-2-yl)phenyl(ISO)

230-253-46988-21-2

T;N-methylcarbamate

N;R25R51-53

T;R:N

S:(1/2-)37-45-61

25-51/53

006-033-00-7metoxuron3-(3-chloro-4-methoxyphenyl)-1,1-(ISO)

243-433-219937-59-8N;R50-53

N

dimethylurea

R:S:60-6150/53006-034-00-2pebulateN-butyl-N(ISO)

-ethyl-S-propylthiocarbamate

214-215-41114-71-2

Xn;N;R51-53

R22Xn;R:N

S:(2-)23-61

22-51/53006-035-00-8

pirimicarb5,6-dimethyl-2-dimethylamino-pyrimidin-(ISO)

245-430-123103-98-2

T;T;4-yl-N,N-dimethylcarbamateN;R25R50-53

R:N

S:(1/2)22-37-45-60-6125-50/53

006-037-00-9promecarb3-isopropyl-5-methylphenyl(ISO)

220-113-02631-37-0

T;T;N-methylcarbamate

N;R25R50-53

R:N

S:(1/2-)24-37-45-60-6125-50/53

006-038-00-4sulfallate2-chlorallyl(ISO)

N,N-dimethyldithiocarbamate

E

202-388-995-06-7

Carc.Xn;Cat.2;R45T;N

N;R50-53R22R:S:53-45-60-6145-22-50/53006-039-00-X

tri-allateS-2,3,3-trichlorallyl(ISO)

diisopropylthiocarbamate

218-962-72303-17-5

Xn;R43

R22-48/22Xn;N;R50-53R:N

S:(2-)24-37-60-6122-43-48/22-50/53006-042-00-6

monuron3-(4-chlorphenyl)-1,1-dimethylurea

(ISO)

205-766-1150-68-5

Carc.Xn;Cat.3;R40Xn;N;R50-53R22R:S:(2-)36/37-60-6122-40-50/53N

006-043-00-1

monuron-TCA

3-(4-chlorophenyl)-1,1-dimethyluronium󰂗140-41-0

Xi;Xn;trichloroacetate

Carc.R36/38

N;R50-53

Cat.3;R40R:N

S:(2-)36/37-60-61

36/38-40-50/53L136/6ENOfficialJournaloftheEuropeanCommunities8.6.2000IndexNoChemicalname

substances

relatedNotestoECNoCASNoClassificationLabellingConcentrationlimits

Notespreparations

relatedto006-045-00-2

methomyl1-(methylthio)ethylideneamino(ISO)

240-815-016752-77-5

T+;N-methylcarbamate

N;R50-53

R28T+;R:N

S:(1/2-)22-36/37-45-60-61

28-50/53

006-046-00-8bendiocarb2,2-dimethyl-1,3-benzodioxol-4-yl(ISO)

245-216-822781-23-3

T;N-methylcarbamate

Xn;R23/25T;N;R50-53R21R:N

S:(1/2-)22-36/37-45-60-6121-23/25-50/53

006-047-00-3bufencarbA󰂗8065-36-9

T;Nmixture(ISO)

of3-(1-methylbutyl)phenylN;R24/25R50-53

T;R:N

3-(1-ethylpropyl)phenyl-methylcarbamateand

N-methylcarbamateS:(1/2-)28-36/37-45-60-61

24/25-50/53

006-048-00-9ethiofencarb2-(ethylthiomethyl)phenyl(ISO)

N-methylcarbamate

249-981-929973-13-5

Xn;N;R50-53

R22Xn;R:S:(2-)60-6122-50/53N

006-050-00-X

fenuron-TCA

1,1-dimethyl-3-phenyluroniumtrichloroacetate

󰂗4482-55-7

Xi;N;R50-53

R38Xi;R:N

S:(2-)60-6138-50/53006-053-00-6

isoprocarb2-isopropylphenyl(ISO)

N-methylcarbamate

220-114-62631-40-5

Xn;N;R50-53

R22Xn;R:N

S:(2-)60-61

22-50/53006-054-00-1

mexacarbate3,5-dimethyl-4-dimethylaminophenyl(ISO)

206-249-3315-18-4

T+;T+;N-methylcarbamate

Xn;R28N;R50-53R21R:S:(1/2-)36/37-45-60-6121-28-50/53

N

006-057-00-8nitrapyrin2-chloro-6-trichloromethylpyridine

(ISO)

217-682-21929-82-4

Xn;N;R51-53

R22Xn;R:N

S:(2-)24-6122-51/53006-060-00-4

oxycarboxin2,3-dihydro-6-methyl-5-((ISO)

226-066-25259-88-1

Xn;Xn

1,4-oxothiine4,4-dioxideN-phenylcarbamoyl)-R52-53

R22R:S:(2-)61

22-52/53006-069-00-3thiophanate-methyl1,2-di-(3-methoxycarbonyl-(ISO)245-740-7235-05-8

Muta.Xn;2-thioureido)benzene

N;R50-53

Cat.3;R40R:N

S:(2-)36/37-60-6140-50/53

006-070-00-9furmecyclox

N262-302-060568-05-0

Carc.Xn;3-furamide

-cyclohexyl-N-methoxy-2,5-dimethyl-N;R50-53

Cat.3;R40R:N

S:(2-)36/37-60-61

40-50/53

8.6.2000ENOfficialJournaloftheEuropeanCommunitiesL136/7IndexNoChemicalname

substances

relatedNotestoECNoCASNoClassificationLabellingConcentrationlimits

Notespreparations

relatedto006-088-00-7

benfuracarbethyl󰂗82560-54-1

T;7-yloxycarbonyl(methyl)aminothio]-N-[2,3-dihydro-2,2-dimethylbenzofuran-(ISO)

N;R23/25R50-53

T;R:N

N-isopropyl-b-alaninateS:(1/2-)36/37-45-60-61

23/25-50/53

007-012-00-5N1,1-dimethylhydrazine

,N-dimethylhydrazineE

200-316-057-14-7

F;Carc.R11

F;T;Cat.2;R45R:T;N

C;R23/25S:53-45-61

45-11-23/25-34-51/53N;R34R51-53007-013-00-0

N1,2-dimethylhydrazine

,N-dimethylhydrazineE󰂗540-73-8

Carc.T;Cat.2;R45T;N;R23/24/25R51-53

R:N

45-23/24/25-51/53CR45-23/24/25Å25%:T;S:53-45-61

3%R45-20/21/22

ÄC<25%:T;0,01%ÄC<3%:T;R45009-003-00-1hydrofluoricacid󰂅%B231-634-876-39-3

T+;C;R35

R26/27/28T+;R:C

CS:(1/2-)7/9-26-36/37-45

26/27/28-35

R26/27/28-35Å7%:T+;C;1%R23/24/25-34ÄC<7%:T;0,1R20/21/22-36/37/38

%ÄC<1%:Xn;015-039-00-9

azinphos-methylO,O-dimethyl4-oxobenzotriazin-3-(ISO)

201-676-186-50-0

T+;T+;ylmethylphosphorodithioate

T;R26/28R43

R24R:N

N;R50-53S:(1/2-)28-36/37-45-60-61

24-26/28-43-50/53

015-048-00-8

fenthionO,O-dimethyl-(ISO)

O-(4-methylthio-m200-231-955-38-9

Muta.T;Cat.3;R40T;tolyl)phosphorothioate

-Xn;R23-48/25R:N

N;R50-53R21/22S:(1/2-)36/37-45-60-61

21/22-23-40-48/25-50/53015-056-00-1

azinphos-ethylO,O-diethyl(ISO)

220-147-622-71-9

T+;T;R28T+;ylmethylphosphorodithioate

4-oxobenzotriazin-3-N;R24R50-53R:N

S:(1/2-)28-36/37-45-60-6124-28-50/53

015-140-00-8triazophosO245-986-524017-47-8

T;Xn;R23/25T;3-ylphosphorothioate,O-diethyl-(ISO)

O-1-phenyl-1H,2,4-triazol-N;R50-53R21R:N

S:(1/2-)36/37-45-60-6121-23/25-50/53

016-013-00-Xsulphurdichloride

234-129-010545-99-0

R14C;C;N;R34R50

R:N

14-34-50

CS:(1/2-)26-36/37/39-45-61

5%Å10R36/37/38

ÄC%:<10C;%:R34Xi;L136/8ENOfficialJournaloftheEuropeanCommunities8.6.2000IndexNoChemicalname

substances

relatedNotestoECNoCASNoClassificationLabellingConcentrationlimits

Notespreparations

relatedto016-014-00-5sulphurtetrachloride󰂗13451-08-6

R14C;R34C;N;R50

R:N

14-34-50

CS:(1/2-)26-36/37/39-45-615%Å10R36/37/38

ÄC%:<10C;%:R34Xi;016-023-00-4dimethylsulphateE201-058-177-78-1

Carc.Muta.Cat.2;R45T+

CT+;Cat.3;R40R:R45-25-26-34-43Å25%:T+;

T;R25R26S:53-45

45-25-26-34-4310C;R45-22-26-34-43%ÄC<25%:T+;R43

R347%R45-22-26-36/37/38-43ÄC<10%:T+;

5%R45-22-23-36/37/38-43ÄC<7%:T;

3R45-22-23-43%ÄC<5%:T;1%R45-23-43

ÄC<3%:T;0,1R45-20

%ÄC<1%:T;0,01R45

%ÄC<0,1%:T;016-024-00-X

dimexanobis(methoxythiocarbonyl)(ISO)

disulphide

215-993-81468-37-7

Xn;N;R50-53

R22Xn;R:N

S:(2-)60-6122-50/53016-071-00-6

trisodium-3-amino-6,13-dichloro-10-((3-((4-chloro-6-(2-sulfophenylamino)-410-130-3136248-03-8R43

Xi1,3,5-triazin-2-yl)amino)propyl)amino]-4,11-R:triphenoxydioxazinedisulfonateS:(2-)22-24-37

43

022-001-00-5titaniumtetrachloride

231-441-97550-45-0

R14C;R34

C

R:CS:(1/2-)7/8-26-36/37/39-4514-34

5%Å10%:C;R34R36/37/38

ÄC<10%:Xi;030-004-00-8

dimethylzincdiethylzinc[2][1]208-884-1209-161-3[1][2]544-97-8557-20-0[1][2]

R14F;F;C;R17R:C;N

N;R34R50-53S:(1/2-)16-43-45-60-61

14-17-34-50/53

050-002-00-0

cyhexatinhydroxytricyclohexylstannane(ISO)

236-049-113121-70-5

Xn;N;R20/21/22Xn;tri(cyclohexyl)tinhydroxide

R50-53

R:N

S:(2-)13-60-61

20/21/22-50/538.6.2000ENOfficialJournaloftheEuropeanCommunitiesL136/9IndexNoChemicalname

substances

relatedNotestoECNoCASNoClassificationLabellingConcentrationlimits

Notespreparations

relatedto050-012-00-5

tetracyclohexylstannanechlorotricyclohexylstannane[1][2]215-910-51449-55-4Xn;Å1%:Xn;R20/21/221

butyltricyclohexylstannane[3]

221-437-5[1]230-358-5[2][3]3091-32-5[1]7067-44-9[2][3]N;R50-53

R20/21/22Xn;R:N

CS:(2-)26-28-60-6120/21/22-50/53050-017-00-2

fenbutatinbis(tris(2-methyl-2-phenylpropyl)tin)oxide

oxide(ISO)

236-407-7

13356-08-6

T+;Xi;R26T+;N;R50/53R36/38R:S:(1/2-)28-36/37-45-60-6126-36/38-50/53

N

082-009-00-X

leadC.I.215-693-71344-37-2Carc.Repr.Cat.3;R40T;1

identifiedPigmentsulfochromateConstitutioninYellowyellow

theNumber,Colour34[ThisC.I.Indexsubstance77603.]

byColouris

IndexRepr.Cat.1;R61R:N

R33

Cat.3;R62S:53-45-60-61

61-33-40-50/53-62N;R50-53082-010-00-5

leadC.I.Pigmentchromateredmolybdatesulfatered235-759-912656-85-8

Carc.Repr.Cat.T;identifiedRepr.Cat.3;1;R40R:N

1

ConstitutionintheNumber,Colour104.[Thissubstanceis

C.I.Index77605.]

byColourIndexR33

Cat.3;R61R62S:53-45-60-61

61-33-40-50/53-62N;R50-53601-024-00-X

cumenepropylbenzene[1]

[2]202-704-5203-132-9[1][2]103-65-198-82-8[1][2]

R10Xn;Xn;Xi;R65R:10-37-51/53-65N

4

N;R51-53R37S:(2-)24-37-61-62

601-032-00-3

benzo[benzo[adef]pyrene]chrysene

200-028-550-32-8

Carc.Muta.Cat.2;T;Repr.R:N

R60-61Cat.Cat.2;2;R45R46S:53-45-60-61

45-46-60-61-50/53N;R50-53601-034-00-4benz[e]acephenanthrylene205-911-9205-99-2

Carc.N;R50-53

Cat.2;R45T;R:N

S:53-45-60-6145-50/53602-035-00-2

1,4-dichlorobenzenep-dichlorobenzene

203-400-5106-46-7

Xi;N;R50-53

R36Xi;R:S:(2-)24/25-46-60-6136-50/53

N

602-054-00-6

3-iodopropeneallyliodide

209-130-4556-56-9

R10C;R34

C

R:S:(1/2-)7-26-45

10-34

L136/10ENOfficialJournaloftheEuropeanCommunities8.6.2000IndexNoChemicalname

substances

relatedNotestoECNoCASNoClassificationLabellingConcentrationlimits

Notespreparations

relatedto603-076-00-9

but-2-yne-1,4-diol2-butyne-1,4-diol

203-788-6110-65-6

T;Xn;R23/25T

CC;R34R21-48/22R:S:21-23/25-34-48/22R21-23/25-34-48/22Å50%:T;

(1/2-)26-36/37/39-45

25R21-23/25-36/38-48/22%ÄC<50%:T;

10R20/22-48/22%ÄC<25%:Xn;3R20/22

%ÄC<10%:Xn;603-091-00-0

exo-1-methyl-4-(1-methylethyl)-7-oxabi-cyclo[2.2.1]heptan-2-ol

402-470-687172--2

O;Xn;R8O;Xi;R36R22R:XnS:(2-)26

8-22-36603-093-00-1

exo-(+/-)-1-methyl-4-(1-methylethyl)-2-[(2-methylphenyl)methoxy]-7-oxabi-402-410-987818-31-3

Xn;N;R51-53

R20Xn;cyclo[2.2.1]heptaneR:S:(2-)23-6120-51/53N

603-097-00-31,1triisopropanolamine

¡,1¢-nitrilotripropan-2-ol204-528-4122-20-3

Xi;R52-53

R36Xi

R:S:(2-)26-6136-52/53603-117-00-0

propan-2-ol

isopropyl200-661-767-63-0

F;F;R:Xi

6

isopropanolalcoholXi;R11R67

R36S:(2-)7-16-24/25-2611-36-67

604-020-00-6biphenyl-2-ol

2-hydroxybiphenyl201-993-590-43-7

Xi;R50

Xi;2-phenylphenol(ISO)N;R36/37/38R:N

S:(2-)22-6136/37/38-50604-021-00-12-phenylphenol,sodium2-biphenylate

sodiumsalt205-055-6132-27-4

Xn;Xi;R22

Xn;N;R50

R37/38-41R:N

S:(2-)22-26-6137/38-41-50604-024-00-8

4,4(alt.):¡-isobutylethylidenediphenol

2,2-bis(4¡hydroxyphenyl)-4-methylpentane401-720-16807-17-6

Repr.Xi;Cat.2;R60T;N;R50-53R36R:N

S:53-45-60-6160-36-50/53604-041-00-0

acifluorfenacifluorfen-sodium[1]

[2]

256-634-5Xn;Xn;5-[2-chloro-4-(trifluormethyl)phenoxy]-263-560-7[1][2]50594-66-662476-59-9[1][2]

Xi;R22R:N

2-nitrobenzoicN;R50-53

R38-41S:(2-)24-39-60-61

22-38-41-50/53sodium-5-[2-chloro-4-(trifluormethyl)acid[1]

phenoxy]-2-nitrobenzoate[2]

8.6.2000ENOfficialJournaloftheEuropeanCommunitiesL136/11IndexNoChemicalname

substances

relatedNotestoECNoCASNoClassificationLabellingConcentrationlimits

preparations

relatedNotesto604-043-00-1

monobenzone

4-hydroxyphenyl203-083-3103-16-2

Xi;Xi

hydroquinonemonobenzylbenzyletheretherR43R36R:S:(2-)24/25-26-37

36-43

604-044-00-7mequinol

4-methoxyphenol

205-769-8150-76-5

Xn;Xn

hydroquinonemonomethyletherXi;R22R43

R36R:S:(2-)24/25-26-37/39-4622-36-43

605-016-00-7glyoxalethandial󰂅󰂅%%

B

203-474-9107-22-2

Muta.Xn;Cat.Xn

Xi;R203;R40R:CR43R36/38S:(2-)36/3720-36/38-40-43R20-36/38-40-43Å10%:Xn;

1%R40-43

ÄC<10%:Xn;606-016-00-X

pindone2-pivaloylindan-1,3-dione(ISO)

201-462-883-26-1

T;N;R25-48/25R50-53T;R:N

S:(1/2-)37-45-60-6125-48/25-50/53606-018-00-0dichlone2,3-dichloro-1,4-naphthoquinone(ISO)

204-210-5117-80-6

Xn;Xi;Xn;N;R50-53R36/38R22R:S:(2-)26-60-61

22-36/38-50/53N

606-019-00-6chlordeconeperchloropentacyclo[5,3,0,0(ISO)

2,6205-601-3143-50-0

Carc.decan-5-one

,03,9,04,8]T;T;decachloropentacyclo[5,2,1,02,6N;R24/25Cat.3;R40R50-53

R:N

S:(1/2-)22-36/37-45-60-61

24/25-40-50/53

decan-4-one

,03,9,05,8]606-034-00-8metribuzin4-amino-6-(ISO)

244-209-721087--9

Xn;triazin-5(4H)-one

tert-butyl-3-methylthio-1,2,4-N;R50-53

R22Xn;R:N

4-amino-4,5-dihydro-6-(1,1-dimethylethyl)-S:(2-)60-61

22-50/533-methylthio-1,2,4-triazin-5-one

606-035-00-3chloridazon5-amino-4-chloro-2-phenylpyridazine-3-(2H)-one(ISO)

216-920-21698-60-8

R43

pyrazon

N;R50-53Xi;R:N

S:(2-)24-37-60-61

43-50/53

606-036-00-9quinomethionatechinomethionat219-455-32439-01-2

Repr.6-methyl-1,3-dithiolo(4,5-b)quinoxalin-2-one

(ISO)

Xn;

Cat.3;R62Xn;R20/21/22-48/22R:N

Xi;53-62

20/21/22-36-43-48/22-50/R43

R36S:(2-)24-37-60-61

N;R50-53606-037-00-4

triadimefon1-(4-chlorphenoxy)-3,3-dimethyl-(ISO)

256-103-843121-43-3

Xn;N;R51-53

R22Xn;1-(1,2,4-triazol-1-yl)butanone

R:N

S:(2-)61

22-51/53L136/12ENOfficialJournaloftheEuropeanCommunities8.6.2000IndexNoChemicalname

substances

relatedNotestoECNoCASNoClassificationLabellingConcentrationlimits

Notespreparations

relatedto606-044-00-22,4,6-trimethylbenzophenone403-150-9954-16-5

Xn;Xi;R36R22Xn;N;R50-53R:S:(2-)26-60-6122-36-50/53N

607-043-00-X

dicamba2,5-dichloro-6-methoxybenzoic(ISO)

217-635-61918-00-9

Xn;3,6-dichloro-2-methoxybenzoicacidacidXi;R22Xn;R52-53R41R:N

S:(2-)26-6122-41-52/53607-057-00-6coumachlor3-[1-(4-chlorphenyl)-3-oxobutyl)-4-hydroxy-(ISO)

201-378-181-82-3

Xn;coumarin

R52-53R48/22Xn

R:S:(2-)37-6148/22-52/53607-058-00-1cuomafurylfumarin

(ISO)204-195-5117-52-2

T;R52-53

R25-48/25T

(RS)-3-(1-(2-furyl)-3-oxobutyl)-R:4-hydroxycoumarin

S:(1/2-)37-45-61

25-48/25-52/534-hydroxy-3-[3-oxo-1-(2-furyl)butyl]coumarin607-079-00-6kelevanethyl-5-(perchloro-5-hydroxypentacyclo

(ISO)

󰂗4234-79-1

T;(5,3,0,02,6Xn;R24T;ethyl-5-(1,2,3,5,6,7,8,9,10,10-decachloro-4-,03,9,04,8)decan-5-yl)-4-oxopentanoateN;R51-53R22R:N

S:(1/2-)36/37-45-61

22-24-51/53

hydroxypentacyclo(5,2,1,02,6yl)-4-oxovalerate

,03,9,05,8)dec-4-607-097-00-4benzene-1,2,4-tricarboxylictrimelliticanhydrideacid1,2-anhydride209-008-0552-30-7

Xi;R42/43R37-41Xn

R:S:(2-)22-26-36/37/3937-41-42/43

607-143-00-3valericacid

203-677-2109-52-4

C;R52-53R34C

R:S:(1/2-)26-36-45-6134-52/53

607-152-00-2

2,3,6-TBA2,3,6-trichlorobenzoic(ISO)

acid

200-026-450-31-7

Xn;N;R51-53R22Xn;R:S:(2-)6122-51/53N

607-153-00-8benazolin4-chloro-2,3-dihydro-2-oxo-1,3-benzothiazol-(ISO)

223-297-03813-05-6

Xi;R52-53R36/38Xi

3-ylaceticacid

R:S:(2-)22-6136/38-52/53607-156-00-4chlorfenson4-chlorophenyl-4-chlorobenzenesulfonate(ISO)

201-270-480-33-1

Xn;Xi;R22Xn;N;R50-53R38R:S:(2-)37-60-6122-38-50/53N

607-158-00-5sodiumsodiumsaltchloroacetate

ofchloroaceticacid223-498-33926-62-3

T;Xi;R25T;N;R50

R38R:N

S:(1/2-)22-37-45-61

25-38-50

8.6.2000ENOfficialJournaloftheEuropeanCommunitiesL136/13IndexNoChemicalname

substances

relatedNotestoECNoCASNoClassificationLabellingConcentrationlimits

preparations

relatedNotesto607-159-00-0

chlorobenzilateethyl(ISO)

208-110-2510-15-6

Xn;ethyl-4,42,2-di(4-chlorophenyl)-2-hydroxyacetate¡-dichlorobenzilate

N;R50-53

R22Xn;R:N

S:(2-)60-61

22-50/53607-176-00-3A5-tert-butyl-4-hydroxyphenyl)propionyl-mixtureof:a-3-(3-(2H-benzotriazol-2-yl)-400-830-7󰂗

R43

xN;R51-53

Xi;R:N

benzotriazol-2-yl)-5-tert-butyl-4--hydroxypoly(oxyethylene);a-3-(3-(2H-S:(2-)36/37-61

43-51/53hydroxyphenyl)propionyl-benzotriazol-2-yl)-5-tert-butyl-x-3-(3-(2H-4-hydroxyphenyl)propionyloxypoly(oxyethylene)

607-188-00-9hydrogenN-carboxylatoethyl-N-octadec-9-enylmaleamate

sodium

402-970-4󰂗

R43

N;R51-53

Xi;R:N

S:(2-)24/37-6143-51/53607-209-00-1

AO,O-di(1-methylethyl)trithio-bis-thioformate;mixtureof:

403-030-6󰂗

Xn;R43

R22Xn;O,O-di(1-methylethyl)tetrathio-bis-thio-R:N

formate;O,O-di(1-methylethyl)pentathio-bis-N;R50-53

S:(2-)36/37-60-61

22-43-50/53thioformate

607-213-00-3ethyl-3,3-bis[(1,1-dimethylpropyl)peroxy]butyrate

403-320-267567-23-1

E;O;R2R7E;R10

R:N

N;R51-53S:(2-)3/7-14-33-36/37/39-61

2-7-10-51/53

607-217-00-5

2-ethoxyethyl-2-[4-(2,6-dihydro-2,6-dioxo-7-phenyl-1,5-dioxaindacen-403-960-2󰂗

R43Xi

3-yl)phenoxy]acetate

R53

R:S:(2-)24-37-6143-53

607-243-00-7

sodium3,6-dichloro-3,6-dichloro-o217-846-32,2¡-iminodiethanolo-anisic(1:1)acid,-anisatecompound[1]

with246-590-5[1][2]25059-78-31982-69-0[1]R52-53

R:258-527-9[3]53404-28-7[2][3]

S:61

52/533,6-dichloro-2-aminoethanolo-anisic(1:1)acid,[2]

[3]compoundwith607-248-00-4naptalam-sodium

sodiumN-naphth-1-ylphthalamate

205-073-4132-67-2Xn;R22

XnR:S:(2)

22L136/14ENOfficialJournaloftheEuropeanCommunities8.6.2000IndexNoChemicalname

substances

relatedNotestoECNoCASNoClassificationLabellingConcentrationlimits

preparations

relatedNotesto607-249-00-X

(1-methyl-1,2-ethanediyl)bis[oxy(methyl-2,1-ethanediyl)diacrylate

256-032-242978-66-5

Xi;R43

R36/37/38Xi;CtripropyleneTPGDA

glycoldiacrylateN;R51-53R:N

S:(2-)24-37-61

36/37/38-43-51/53R36/37/38-43

Å10%:Xi;1%ÄC<10%:Xi;R43

607-252-00-6lambda-cyhalothrina415-130-791465-08-6

T+;(Z)-(1R)-1:1mixture(ISO)

T;R26T+;2,2-dimethylcyclopropanecarboxylatecis-3-(2-chloro-3,3,3-trifluoropropenyl)-of(S)-a-cyano-3-phenoxybenzylXn;R25R:N

(N;R50-53

R21S:

21-25-26-50/53(1/2-)28-36/37/39-38-45-60-61

3-(2-chloro-3,3,3-trifluoropropenyl)-R)-a-cyano-3-phenoxybenzyl(Z)-(1S)-andcis-2,2-dimethylcyclopropanecarboxylate607-255-00-2fluroxypyr4-amino-3,5-dichloro-6-fluoro-(ISO)

󰂗69377-81-7R52-53

2-pyridyloxyaceticacidR:S:61

52/53608-003-00-4acrylonitrile

DE

203-466-5107-13-1

F;Carc.R11

F;T;Cat.2;R45R:T;45-11-23-/24/25-37/38-41-N

CR45-23/24/25-37/38-Å20%:T;

Xi;R23/24/25R43

R37/38-41S:9-16-53-45-61

43-51/53

41-43

10%ÄN;R51-53

R45-23/24/25-41-43C<20%:T;5%R45-23/24/25-36-43ÄC<10%:T;1%R45-23/24/25-43ÄC<5%:T;0,2R45-20/21/22

%ÄC<1%:T;0,1%ÄC<0,2%:T;R45

608-016-00-51,4-Dicyano-2,3,5,6-tetra-chloro-benzene401-550-817-41-2

R43

N;R50-53Xi;R:N

S:(2-)24-37-60-6143-50/53

609-030-00-4

dinoterb2-tert-butyl-4,6-dinitrophenol

(ISO)

E215-813-81420-07-1

Repr.T+;Cat.2;R61T+;T;R28R:R44

R24S:53-45-60-61

61-24-28-44-50/53N

N;R50-53609-040-00-9

nitrofen2,4-dichlorophenyl(ISO)

4-nitrophenylether

E217-406-01836-75-5

Carc.Repr.Cat.T;Xn;Cat.2;2;R45R61R:N

N;R50-53R22S:53-45-60-6145-61-22-50/53609-044-00-0

tecnazene1,2,4,5-tetrachloro-3-nitrobenzene

(ISO)

204-178-2117-18-0

Xn;R43

R22Xn;N;R50-53

R:N

S:(2-)24-37-60-61

22-43-50/538.6.2000ENOfficialJournaloftheEuropeanCommunitiesL136/15IndexNoChemicalname

substances

relatedNotestoECNoCASNoClassificationLabellingConcentrationlimits

preparations

relatedNotesto611-008-00-4

4-aminoazobenzene4-phenylazoaniline

200-453-660-09-3

Carc.N;R50-53

Cat.2;R45T;R:N

S:53-45-60-61

45-50/53611-013-00-1

trilithium2-(3-methyl-4-(2-methoxy-4-1-hydroxy-7-(3-sulfonatoanilino)-403-650-7117409-78-6

E;(3-sulfonatophenylazo)phenylazo)phenylazo)N;R2

R51-53

E;R:N

naphthalene-3-sulfonate

S:(2-)35-61

2-51/53611-031-00-X4,4ene)dianiline¡-(4-iminocyclohexa-2,5-dienylidenemethyl-209-321-2569-61-9Carc.Cat.2;R45

TC.I.BasicRedhydrochloride9R:S:53-4545612-035-00-42-methoxyanilineo-anisidine

E

201-963-190-04-0

Carc.Muta.Cat.T

T;R23/24/25Cat.2;3;R45R40R:S:53-4545-23/24/25612-042-00-2

benzidine

1,1E202-199-192-87-5

Carc.4,4¡-byphenyl-4,4¡-diamineXn;Cat.1;R45T;biphenyl-4,4¡-diaminobiphenyl

¡-ylenediamineN;R50-53

R22R:N

C0,01Å25%%:S:53-45-60-61

45-22-50/53ÄCT;<25R45-22

%:T;R45

612-051-00-14,44,4¡¡-diaminodiphenylmethane-methylenedianiline

E202-974-4101-77-9

Carc.Muta.Cat.T;T;Cat.2;R45R:N

45-39/23/24/25-43-48/20/R48/20/21/22R39/23/24/25Xn;3;R40R43

S:53-45-61

21/22-51/53N;R51-53612-081-00-5

saltssaltsof4,4¡-bi-ACarc.saltsofof3,3o-tolidine

¡-dimethylbenzidineo-toluidine

E

210-322-5265-294-7Xn;Cat.2;R45T;277-985-0969-36-4612-82-874753-18-7

N;R51-53R22R:N

S:53-45-61

45-22-51/53612-099-00-34-methyl-2,4-toluenediamine

m-phenylenediamineE

202-453-1

95-80-7

Carc.T;Cat.2;R45T;Xn;R25R:N

Xi;R21S:53-45-61

45-21-25-36-43-51/53R43

R36N;R51-53612-105-00-42-piperazin-1-ylethylamine205-411-0140-31-8

Xn,C;R21/22C

R43R34R:R52-53

S:(1/2-)26-36/37/39-45-61

21/22-34-43-52/53

L136/16ENOfficialJournaloftheEuropeanCommunities8.6.2000IndexNoChemicalname

substances

relatedNotestoECNoCASNoClassificationLabellingConcentrationlimits

Notespreparations

relatedto612-111-00-7

2-methyl-2,6-toluenediamine

m-phenylenediamine212-513-9823-40-5

Muta.Xn;Cat.3;R40Xn;R43

R21/22R:N

N;R51-53S:(2-)24-36/37-61

21/22-40-43-51/53612-125-00-3

2-methyl-2,5-toluenediamine

p-phenylenediamine202-442-195-70-5

T;Xn;R25T;R43

R20/21R:N

N;R51-53S:(1/2-)24-37-45-61

20/21-25-43-51/53612-144-00-7

flumetralinN-(2-chloro-6-fluorobenzyl)-(ISO)

N-ethyl-a,a,󰂗62924-70-3

Xi;Xi;trifluoro-2,6-dinitro-p-toluidinea-R43

R36/38N;R50-53R:N

S:(2-)36/37-60-61

36/38-43-50/53612-151-00-5diaminotoluene

E

246-910-325376-45-8

Carc.T;Cat.T;Xn;R252;R45R:N

Xi;S:53-45-61

45-20/21-25-36-43-51/53R43

R36R20/21N;R51-53613-018-00-4

morfamquat1,1󰂗7411-47-4

Xn;Xi;Xn

methyl)-4,4¡-bis(3,5-dimethylmorpholinocarbonyl-(ISO)

¡-bipyridiliumionR52-53R36/37/38R22

R:S:(2-)22-36-6122-36/37/38-52/53613-031-00-5symclosene

trichloroisocyanuricacid201-782-887-90-1

O;Xn;R8O;trichloro-1,3,5-triazinetrion

R31

R22R:Xn;N

Xi;S:(2-)8-26-41-60-61

8-22-31-36/37-50/53N;R50-53R36/37613-038-00-3

6-phenyl-1,3,5-triazine-2,4-diyldiamine6-phenyl-1,3,5-triazine-2,4-diamine202-095-691-76-9

Xn;R52-53

R22Xn

benzoguanamine

R:S:(2-)61

22-52/53613-042-00-5imazalil1-[2-(allyloxy)-2-(2,4-dichlorophenyl)ethyl]-(ISO)

252-615-035554-44-0

Xn;1H-imidazole

N;R20/22Xn;N;R41R50-53R:N

S:(2-)26-39-60-6120/22-41-50/53613-043-00-0

imazalil1-[2-(allyloxy)ethyl-2-(2,4-dicholorphenyl)]-sulphate(ISO)

261-351-51281-291-3[1][2]58594-72-283918-57-4[1][2]

Xn;Xi;R20/22Xn;(±)-1-[2-(allyloxy)ethyl-2-(2,4-dichlorophenyl)]-H-imidazoliumhydrogensulphate[1]

N;R50-53

R41R:N

S:(2-)26-39-60-61

20/22-41-50/531H-imidazoliumhydrogensulphate[2]

8.6.2000ENOfficialJournaloftheEuropeanCommunitiesL136/17IndexNoChemicalname

substances

relatedNotestoECNoCASNoClassificationLabellingConcentrationlimits

preparations

relatedNotesto613-066-00-6

terbumeton2-(ISO)

251-637-833693-04-8

Xn;1,3,5-triazin

tert-butylamino-4-ethylamino-6-methoxy-N;R50-53

R22Xn;R:N

S:(2-)60-61

22-50/53613-091-00-2morfamquatmorfamquatdichloridesulfate[2]

[1]225-062-8[1]

29873-36-74636-83-3[1][2]

Xn;Xi;R22

Xn;

R52-53R36/37/38R:S:(2-)22-36-61

22-36/37/38-52/53613-098-00-0N-(n-octyl)-2-pyrrolidinone403-700-82687-94-7

C;N;R34R51-53

C;R:N

S:(1/2-)23-26-36/37/39-45-6134-51/53

613-130-00-3

hexaconazole(RS)-2-(2,4-dichlorophenyl)-1-(1(ISO)

H󰂗79983-71-4

R43

Xi;triazol-1-yl)hexan-2-ol

-1,2,4-N;R51-53

R:S:(2-)24-37-6143-51/53N

613-131-00-9pyroquilon1,2,5,6-tetrahydropyrrolo[3,2,1-(ISO)

󰂗57369-32-1

Xn;R52-53

R22Xn

4-one

ij]quinolin-R:S:(2-)61

22-52/53613-134-00-5myclobutanil2-(4-chlorophenyl)-2-(1(ISO)

󰂗88671--0

Repr.Xn;1-ylmethyl)hexanenitrile

H-1,2,4-triazol-Xn;Cat.3;R63Xi;R:N

N;R51-53R36R22S:(2-)36/37-46-61

22-36-51/53-63613-137-00-1

methabenzthiazuron1-(1,3-benzothiazol-2-yl)1,3-dimethylurea

(ISO)

242-505-018691-97-9

N;R50-53

N

R:S:60-6150/53613-139-00-2

metsulfuron-methyl

methyl󰂗74223--6N;R50-53

N

2-ylcarbamoylsulfamoyl)2-(4-methoxy-6-methyl-1,3,5-triazin-benzoateR:S:60-61

50/53614-001-00-4nicotine3-(N-methyl-2-pyrrolidinyl)pyridine

(ISO)

200-193-354-11-5

T+;T;T+;N;R25R27R51-53R:N

S:(1/2-)36/37-45-6125-27-51/53

614-006-00-1

brucine

2,3-dimethoxystrychnine

206-614-7357-57-3

T+;R52-53

R26/28T+

R:S:(1/2-)13-45-61

26/28-52/53L136/18ENOfficialJournaloftheEuropeanCommunities8.6.2000IndexNoChemicalname

substances

relatedNotestoECNoCASNoClassificationLabellingConcentrationlimits

preparations

relatedNotesto614-007-00-7

brucinebrucinesulphate[1]225-432-94845-99-2[1]Strychnidin-10-one,nitrate[2]

227-317-9[1]mono[(269-439-5[2]68239-26-95786-97-0[2]T+;R52-53

R26/28T+

R:boxylate]R)-1-methylheptyl2,3-dimethoxy-,

1,2-benzenedicar-269-710-8[3][4]68310-42-9[3][4]

S:(1/2-)13-45-61

26/28-52/53Strychnidin-10-one,[3]

with2,3-dimethoxy-,compd.dicarboxylate(S)-mono(1-methylheptyl-1,2-benzene-(1:1)[4]

615-006-00-4

2-methyl-4-methyl-mC

202-039-0m209-544-5[1]91-08-7[1]Carc.T+

toluene-tolylidenem-phenylene-phenylenediisocyanatediisocyanate[1][2]247-722-4[2][3]271-62-5584-84-9[2]T+;Cat.3;R40[3]

Xi;R26

R:2

toluene2,6-di-isocyanatediisocyanate[3][1]R42/43R36/37/38S:(1/2-)23-36/37-45-61

26-36/37/38-40-42/43-52/53CR26-36/37/38-40-42/43

Å20%:T+;7%toluene-diisocyanate2,4-di-isocyanate[3]

[2]R52-53

R26-40-42/43ÄC<20%:T+;1%R23-40-42/43ÄC<7%:T;0,1R20-42%ÄC<1%:Xn;616-010-00-9tosylchloramidesodium204-854-7127-65-1

Xn;R31R22C

C;R:R42R34S:(1/2-)7-22-26-36/37/39-45

22-31-34-42

616-034-00-X

pyracarbolid3,4-dihydro-6-methyl-2H-pyran-5-carboxanilide(ISO)

246-419-424691-76-7

R52-53

R:S:61

52/53616-035-00-5cymoxanil

2-cyano-N-[(ethylamino)carbonyl]-261-043-057966-95-7

Xn;R43

R22Xn;2-(methoxyimino)acetamide

N;R50-53R:N

S:(2-)36/37-60-6122-43-50/53617-004-00-91,2,3,4-tetrahydro-1-naphthylhydroperoxide

212-230-0771-29-9

O;Xn;R7O;C;R22R:C;N

CN;R34R50-53S:

7-22-34-50/5310Å25%:C;(1/2-)3/7-14-26

5%ÄC<25R22-34%:C;R34󰂖36/37/39-45-60-61R36/37/38

%ÄC<10%:Xi;617-006-00-Xbis(a,a-dimethylbenzyl)peroxide201-279-380-43-3

O;Xi;R7

O;N;R51-53R36/38R:S:(2-)3/7-14-36/37/39-617-36/38-51/53

Xi;N

617-008-00-0

dibenzoylbenzoylperoxyde

peroxide202-327-694-36-0

E;Xi;R2E;R43

R36R:Xi;

S:(2-)3/7-14-36/37/392-36-43

650-007-00-3chlordimeformN(ISO)

Carc.Cat.3;R40Xn;2-(4-Chlor-o-tolyl)-N1,N1-dimethylformamidine228-200-561-98-3

Xn;N;R50-53

R21/22R:N

S:(2-)22-36/37-60-61

21/22-40-50/538.6.2000ENOfficialJournaloftheEuropeanCommunitiesL136/19IndexNoChemicalname

substances

relatedNotestoECNoCASNoClassificationLabellingConcentrationlimits

Notespreparations

relatedto650-008-00-9

drazoxolon4-(2-chlorophenylhydrazone)-3-methyl-(ISO)

227-197-85707-69-7

T;5-isoxazolone

N;R25R50-53T;R:N

S:(1/2-)22-24-36/37-45-60-6125-50/53

650-009-00-4chlordimeformN243-269-119750-95-9

Carc.dimethylformamidinemonohydrochloride¡-(4-chloro-o-tolyl)-hydrochlorideN,N-Xn;Cat.3;Xn;N2-(4-chloro-o-tolyl)-N1,N1N;R50-53

R22R40R:S:(2-)22-36/37-60-61

22-40-50/53

N

dimethylformamidinehydrochloride-650-033-00-5esfenvalerate(S)-a-cyano-3-phenoxybenzyl-((ISO)

S)-󰂗66230-04-4

T;2-(4-chlorophenyl)-3-methylbutyrateR43

R23/25T;N;R50-53R:N

S:(1/2-)24-36/37/39-45-60-61

23/25-43-50/53

650-041-00-9triasulfuron1-[2-(2-chloroethoxy)phenylsulfonyl]-(ISO)

󰂗82097-50-5

N;R50-53

N

3-(4-methoxy-6-methyl-1,3,5󰂖triazin-2-yl)urea

R:S:60-61

50/53L136/20ENOfficialJournaloftheEuropeanCommunities8.6.2000ANNEX1D

IndexNoChemicalnamerelatedNotestoECNoCASNoClassificationLabellingConcentrationlimits

relatedNotessubstances

prepara-totions

006-090-00-8

2-(3-iodoprop-2-yn-1-yloxy)ethylmate

phenylcarba-408-010-088558-41-2

Xn;Xi;R20Xn

R52-53R41R:S:(2-)22-26-39-6120-41-52/53014-016-00-0A1,3-dihex-5-en-1-yl-1,1,3,3-tetramethyldisilox-mixtureof:

406-490-6󰂗

N;R51-53

N

ane;R:iloxane

1,3-dihex-n-en-1-yl-1,1,3,3-tetramethyldis-S:6151/53015-1-00-9calcium-P,P(hydrogenphosphonate)dihydrate

¡-(1-hydroxyethylene)bis400-480-536669-85-9

R52-53R:S:61

52/53015-165-00-4

Athiobis(4,1-phenylene)-S,S,Smixtureof:

¡,S¡-tetraphenyldi-404-986-7

󰂗

Xi;N;R50-53

R41Xi;sulfoniumbishexafluorophosphate;diphenylR:N

(4-phenylthiophenyl)sulfoniumS:(2-)15-26-39-60-61

41-50/53

phate

hexafluorophos-015-166-00-X3,9-bis(2,6-di-tert-butyl-4-methylphenoxy)-2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]410-290-480693-00-1R53

R:undecane

S:6153015-167-00-53-(hydroxyphenylphosphinyl)propanoicacid

411-200-614657--8Xi;R41

XiR:S:(2-)26-3941

601-050-00-1

benzene,C10-13-alkylderivatives

267-051-067774-74-7N;R50

NR:S:6150601-051-00-74-phenylbut-1-ene405-980-7768-56-9

Xi;N;R51-53R38Xi;R:N

S:(2-)37-61

38-51/53602-083-00-4

diphenylderivativepentabromodiphenylether,pentabromo

ether251-084-232534-81-9

Xn;R

R48/21/22Xn;N;R50-53R:N

S:(1/2-)36/37-45-60-6148/21/22-50/53-602-084-00-X1,1-dichloro-1-fluoroethane

404-080-11717-00-6

N;R52-53-59

N

R:S:59-61

52/53-59603-128-00-02-(phenylmethoxy)naphthalene405-490-3613-62-7R53

R:S:61

538.6.2000ENOfficialJournaloftheEuropeanCommunitiesL136/21IndexNoChemicalname

substances

relatedNotestoECNoCASNoClassificationLabellingConcentrationlimits

relatedNotesprepara-totions

603-129-00-61-tert-butoxypropan-2-ol406-180-057018-52-7

R10Xi;R41Xi

R:S:(2-)26-3910-41603-130-00-1

Aamixtureofisomersof:

406-325-8󰂗

Xn;(oxyethylene)

-((dimethyl)biphenyl)-x-hydroxypolyR52-53R22Xn

R:S:(2-)39-6122-52/53603-131-00-7A1-deoxy-1-[methyl-(1-oxododecyl)amino]3:1mixtureof:

407-290-1󰂗

Xi;R41

Xi-D-glucitol;R:cyl)amino]-D-glucitol

1-deoxy-1-[methyl-(1-oxotetrade-S:(2-)26-39

41

603-132-00-22-hydroxymethyl-9-methyl-6-(1-methylethyl)-1,4-dioxaspiro[4.5]decane

408-200-363187-91-7

Xi;R52-53R38-41Xi

R:S:(2-)26-37/39-6138-41-52/53603-133-00-8A3-[(4-amino-2-chloro-5-nitrophenyl)amino]-mixtureof:

408-240-1󰂗

Xn;propane-1,2-diol;3,3R52-53

R22Xn

R:1,4-phenylenediimino)bis(propan-1,2-diol)¡-(2-chloro-5-nitro-S:(2-)22-36-6122-52/53603-134-00-3Atetradecyl,mixtureofsubstituteddodecyl410-450-3󰂗R53

R:producedS:61

53catalystbydiphenyltheFriedelethers.CraftsThereaction.substanceand/orTheisDiphenylisgroups.etherremovedissubstitutedfromthereactionbyC1-C10product.alkylbetweenThe50/50used.

C1alkylandC6.groupsLinearareC12bondedandC14,randomly603-135-00-9bis[[2,2[2-(2-methoxyethoxy)ethoxy]-titanium¡,2¢-nitrilotris[ethanolato]]-1-N,O]bis410-500-4󰂗

Xi;N;R51-53R41Xi;R:N

S:(2-)26-39-6141-51/53603-136-00-43-((4-(bis(2-hydroxyethyl)amino)-2-nitrophe-nyl)amino)-1-propanol

410-910-3104226-19-9

R43R52-53Xi

R:S:(2-)24-37-6143-52/53603-137-00-XA1-deoxy-1-[methyl-(1-oxohexadecyl[amino]-mixtureof:

411-130-6󰂗

Xi;R41

XiD-glucitol;R:cyl)amino]-D-glucitol

1-deoxy-1-[methyl-(1-oxooctade-S:(2-)26-39

41

603-138-00-53-(2,2-dimethyl-3-hydroxypropyl)toluene

alt.):2,2-dimethyl-3-(3-methylphenyl)propanol

403-140-4103694-68-4R52-53

R:S:61

52/53L136/22ENOfficialJournaloftheEuropeanCommunities8.6.2000IndexNoChemicalname

relatedNotessubstances

relatedNotestoECNoCASNoClassificationLabellingConcentrationlimits

prepara-totions

604-050-00-X

4-chloro-4-chloro-2-methylo-cresol

phenol

216-381-31570--5

T;C;R23T;CN;R35R50R:C;N

S:(1/2-)26-36/37/39-45-61

23-35-50

10ÅR20-35

%25Ä%:CÄC<10%:C;3%R20-36/37/38ÄC<5%:Xn;1%R36/37/38

ÄC<3%:Xi;604-051-00-53,5-bis((3,5-di-tert-butyl-4-hydroxy)benzyl)-2,4,6-trimethylphenol

401-110-587113-78-8R52-53R:S:61

52/53604-052-00-02,24-(1,1,3,3-tetramethylbutyl)phenol)¡-methylenebis(6-(2H-benzotriazol-2-yl)-403-800-1103597-45-1R53R:S:61

53604-053-00-6

2-methyl-4-(1,1-dimethylethyl)-6-(1-methyl-pentadecyl)-phenol

410-760-9

157661-93-3

Xi;R43

R38Xi;N;R50-53R:N

S:(2-)24-37-60-6138-43-50/53604-054-00-1A2-methoxy-4-(tetrahydro-4-methylene-mixtureof:

412-020-0󰂗

R432H-pyran-2-yl)-phenol;4-(3,6-dihydro-4-methyl-R52-53

Xi

R:2H-pyran-2-yl)-2-methoxyphenolS:(2-)24-37-6143-52/53604-055-00-72,2-4,4¡-((3,5¡-diyl)-bis(oxymethylene))-bis-oxirane¡,5,5¡-tetramethyl-(1,1¡-biphenyl)413-900-785954-11-6Muta.Cat.3;R40

XnR:S:(2-)22-36-3740

605-027-00-7A3a,4,5,6,7,7a-hexahydro-4,7-methano-1H-in-mixtureof:

410-480-7󰂗

R43

dene-6-carboxaldehyde;3a,4,5,6,7,7a-hexahy-N;R51-53

Xi;R:dro-4,7-methano-1H-indene-5-carboxaldehydeS:(2-)24-37-6143-51/53N

606-051-00-04-pentylcyclohexanone

406-670-461203-83-6N;R51-53

N

R:S:6151/53606-052-00--(N,N-dibutylamino)-2-hydroxy-2¡-carboxybenzophenone

410-410-554574-82-2R52-53R:S:6152/53607-272-00-5

fluoroxypyr-meptylfluroxypyr-butometyl(ISO)279-752-9[1]N

methylheptyl,(ISO)[1][2]

󰂗154486-27-881406-37-3[1]

[2]

N;R50-53

R:fluoro-2-pyridyloxy)O-((4-amino-3,5-dichloro-6-S:60-61

50/532-butoxy-1-methylethyl,acetatedichloro-6-fluoro-2-pyridyloxy)O-(4-amino-3,5-[1]

acetate[2]

8.6.2000ENOfficialJournaloftheEuropeanCommunitiesL136/23IndexNoChemicalname

Notessubstances

relatedtoECNoCASNoClassificationLabellingConcentrationlimits

relatedNotesprepara-totions

607-273-00-0

ammonium-7-(2,6-dimethyl-8-(2,2-dimethylbu-tyryloxy)-1,2,6,7,8,8a-hexahydro-1-naphthyl)-404-520-2󰂗R52-53

R:3,5-dihydroxyheptanoate

S:61

52/53607-274-00-62-(N-benzyl-N-methylamino)ethyl-3-amino-2-butenoate

405-350-154527-73-0

R43

N;R51-53

Xi;R:N

S:(2-)24-37-6143-51/53607-275-00-1sodiumbenzoyloxybenzene-4-sulfonate405-450-566531-87-1R43

XiR:S:(2-)24-37

43

607-276-00-7

bis[(1-methylimidazol)-(2-ethyl-hexanoate)],zinccomplex

405-635-0󰂗

Xi;N;R50-53

R38-41Xi;R:N

S:(2-)26-37/39-60-6138-41-50/53

607-277-00-2

Ahydrochloride;mixtureof:2-(hexylthio)ethylaminesodiumpropionate

405-720-2󰂗

Xn;Xi;R22Xn;R43

R41R:N

N;R51-53S:(2-)24-26-37/39-61

22-41-43-51/53607-278-00-8

Aphenethylnaphthalenesulfonate;mixtureofisomersofsodium

405-760-0󰂗

Xi;Xi

naphthylethylbenzenesulfonate

sodiumR43R41R52-53R:S:(2-)24-26-37/39-6141-43-52/53

607-279-00-3Abis(hydrogenmaleat);mixtureofn-octadecylaminodiethyl

405-960-8󰂗

R43

N;R51-53

Xi;hydrogenmaleatehydrogenphthalaten-octadecylaminodiethylR:N

S:(2-)24-37-6143-51/53607-280-00-9sodium4-chloro-1-hydroxybutane-1-sulfonate

406-190-554322-20-2

Xn;Xi;Xn

R43R36R22R:S:(2-)22-26-36/3722-36-43

607-281-00-4

A3-[3-(2H-benzotriazol-2-yl)-5-(1,1-dimethyl-mixtureofbranchedandlinearC7-C9alkyl407-000-3127519-17-9

N;R51-53

N

ethyl)-4-hydroxyphenyl]propionatesR:S:61

51/53607-282-00-X2-acetoxymethyl-4-benzyloxybut-1-ylacetate

407-140-5131266-10-9R52-53

R:S:61

52/53L136/24ENOfficialJournaloftheEuropeanCommunities8.6.2000IndexNoChemicalname

relatedNotessubstances

relatedNotestoECNoCASNoClassificationLabellingConcentrationlimits

prepara-totions

607-283-00-5E-ethyl-4-oxo-4-phenylcrotonate408-040-415121--8

Xn;Xi;R38-41R21/22Xn;R:N

R43

N;R50-53S:(2-)26-36/37/39-60-61

21/22-38-41-43-50/53607-284-00-0

A3,39:1mixtureof:sodium

410-040-4136213-76-8

N;R51-53

N

panediylimino))bis(10-amino-6,13-dichloro)-¡-(1,4-phenylenebis(carbonylimino-3,1-pro-R:4,11-triphenodioxazinedisulfonate);lithiumS:61

51/533,3propanediyl-imino))bis(10-amino-6,13-dich-¡-(1,4-phenylenebis-(carbonylimino-3,1-loro)-4,11-triphenodioxazinedisulfonate607-285-00-6A7-(((3-aminophenyl)sulfonyl)amino)-naphtha-mixtureof:

410-065-0󰂗R43

Xilene-1,3-disulfonicR:7-(((3-aminophenyl)sulfonyl)amino)-naphtha-acid;sodium

S:(2-)22-24-37

43

lene-1,3-disulfonate;nyl)sulfonyl)amino)-naphthalene-1,3-disulfonatepotassium7-(((3-aminophe-607-286-00-1A7-[[[3-[[4-((2-hydroxy-naphthyl)azo)phe-mixtureof:sodium/potassium

410-070-8141880-36-6

R43nyl]azo]phenyl]sulfonyl]amino]-naphthalene-R52-53

Xi

R:1,3-disulfonate

S:(2-)22-24-37-6143-52/53

607-287-00-7Oethyl)-1,2,3,6-tetrahydrophthalate

¡-methylO-(1-methyl-2-methacryloyloxy-410-140-8󰂗

R52-53R:S:61

52/53607-288-00-2

Tetrasodiumpyrimidin-f-yl(methyl)amino)propyl)-1,6-dihy-(c-(3-(1-(3-(e-6-dichloro-5-cyano-410-160-7

148732-74-5

Xi;dro-2-hydroxy-4-methyl-6-oxo-3-pyridylazo)-R43R36Xi

4-sulfonatophenylsulfamoyl)phtalocyanine-R52-53

R:S:(2-)22-26-36/37-61

36-43-52/53

a,b,d-trisulfonato(6-))nickelatoor(II),whereaor2and16oror317oror4,b18,isd8isor229oror10or11,cisis151andwhere2respectively.

eandftogetherare232orand244oror254607-288-00-83-(3-(4-(2,4-bis(1,1-dimethylpropyl)phen-oxy)butylaminocarbonyl-4-hydroxy-1-naph-410-370-9105488-33-3R53

R:thalenyl)thio)propanoicacid

S:61

53607-290-00-3A1-C14-C18-alkyloxycarbonyl-2-(3-allyloxy-2-hy-mixture(rationotknown)of:ammonium410-540-2󰂗

Xi;droxypropoxycarbonyl)ethane-1-sulfonate;R43

R38Xi;ammoniumN;R50-53

R:N

S:(2-)24-37-60-61

38-43-50/531-(3-allyloxy-2-hydroxypropoxycar-2-C14-C18-alkyloxycarbonyl-bonyl)ethane-1-sulfonate

607-291-00-9dodecyl-x-(C5/C6-cycloalkyl)alkylcarboxylate

410-630-1104051-92-5R53

R:S:61

538.6.2000ENOfficialJournaloftheEuropeanCommunitiesL136/25IndexNoChemicalname

relatedNotessubstances

relatedNotestoECNoCASNoClassificationLabellingConcentrationlimits

prepara-totions

607-292-00-4

A[1-(methoxymethyl)-2-(C12-alkoxy)-mixtureof:

410-0-6󰂗

Xi;ethoxy]aceticN;R50-53

R38-41Xi;R:[1-(methoxymethyl)-2-(C14-alkoxy)-acid;

S:(2-)26-37/39-60-61

38-41-50/53

N

ethoxy]aceticacid

607-293-00-XAmono-2,4,6-trimethylnonyldiphenylmixtureof:N-aminoethylpiperazonium410-650-0󰂗

Xi;di-sulfonate;etherR43

R41Xi;di-2,4,6-trimethylnonyldiphenylN-aminoethylpiperazoniumN;R51-53R:S:(2-)26-36/37/39-61

41-43-51/53

N

di-sulfonate

ether607-294-00-5sodiumate

2-benzoyloxy-1-hydroxyethane-sulfon-410-680-4󰂗R43

XiR:S:(2-)24-3743

607-295-00-0Aphosphonoethane-1,2-dicarboxylate;mixtureof:tetrasodium

410-800-5󰂗

R43

N;R51-53

Xi;diumhexaso-R:N

late

phosphonobutane-1,2,3,4-tetracarboxy-S:(2-)24-37-6143-51/53607-296-00-6Aheptanoicmixtureacidof:pentaerythrioland2-ethylhexanoictetraestersacidwith410-830-9󰂗

R53R:S:6153607-297-00-1

(E-E)-3,3idene)bis(2-oxobornane-10-sulfonic¡-(1,4-phenylenedimethyl-acid)410-960-6

92761-26-7

Xi;R41

XiR:S:(2-)26-3941

607-298-00-72-(trimethylammonium)ethoxycarboxybenzene-4-sulfonate

411-010-3󰂗R43

XiR:S:(2-)22-36/3743

607-299-00-2methyl3-(acetylthio)-2-methyl-propanoate

411-040-797101-46-7

Xn;R43

R22Xn;N;R50-53R:S:(2-)24-37-60-6122-43-50/53N

607-300-00-6

trisodium4-ylamino)-5-(b-sulfamoyl-c,d-sulfonatophthalo-[2-(5-chloro-2,6-difluoropyrimidin-411-430-7󰂗

R43

Xicyanin-a-yl-K4,N29,N30,N31,N32-sulfony-R:lamino)benzoato(5-)]cuprate(II)S:(2-)22-24-37

43

3,22,423,b=24,8,9,25

10,11c=15,16,where17,18da=1,=2,607-301-00-1Aestersmixtureofdodecanoicof:dodecanoicacid

acid;poly(1-7)lactate411-860-5󰂗

Xi;R43

R38-41Xi;N;R:N

R51-53S:(2-)24-26-37/39-61

38-41-43-51/53L136/26ENOfficialJournaloftheEuropeanCommunities8.6.2000IndexNoChemicalname

relatedNotessubstances

relatedNotestoECNoCASNoClassificationLabellingConcentrationlimits

prepara-totions

607-302-00-7

Apoly(1-7)lactatemixtureof:tetradecanoicestersoftetradecanoicacid;

acid411-910-6󰂗

Xi;R43

R38-41Xi;N;R51-53R:S:(2-)24-26-37/39-6138-41-43-51/53N

607-303-00-21-cyclopropyl-6,7-difluoro-1,4-dihydro-4-oxo-quinoline-3-carboxylicacid

413-760-793107-30-3

Repr.R52-53Cat.3;R62Xn

R:S:(2-)22-36/37-6162-52/53

608-023-00-34-(4-chlorophenyl)-2-phenyl-2-[(1H-1,2,4-tria-zol-1-yl)methyl]butanenitrile

406-140-2114369-43-6

N;R50-53

N

R:S:60-61

50/53608-024-00-92-(4-(N-butyl-N-phenethylamino)phenyl)ethy-lene-1,1,2-tricarbonitrile

407-650-7460-76-9R53R:S:6153608-025-00-42-nitro-4,5-bis(benzyloxy)phenylacetonitrile410-970-0117568-27-1

R53

R:S:61

53609-053-00-X

hydrazine-tri-nitromethane

414-850-9

󰂗

E;O;R3E;Carc.R8

R:T

T;Cat.2;R45S:53-45

45-3-8-23/25-43R43

R23/25610-010-00-22-bromo-1-(2-furyl)-2-nitroethylene406-110-935950-52-8

Xn;C;C;R43

R34R22-48/22R:N

N;R50-53S:(1/2-)22-26-36/37/39-45-22-34-43-48/22-50/5360-61611-043-00-5

AN(12:1:1402-850-1

Xi;hydroxy-(or¡)-N(2):N(1mixture£)-N(2of:trisodium

¢)-g-6-[2-amino-4-(or52-53

R41Xi

R:--amino-2-hydroxy)phenylazo]6)-S:(2-)26-39-61

41-52/535¢-(1-carbaniloyl-2-hydroxyprop-1-enylazo)-lene-2,1¡,5£-disulfamoyl-3,3¢-disulfonatobis(naphtha-chromate;¡-azobenzene-1,2-trisodiumN(1¡¡)-N(2):N(1-diolato-O(1),O(2£)-N(2¢)¡))-lazo)-5g-6,6¢-bis(1-carbaniloyl-2-hydroxyprop-1-eny-bis(naphthalene-2,1¡,5£-disulfamoyl-3,3O(1),O(2(1¡))-chromate;¡azobenzene-1,2¢-disulfonato-trisodiumN(1¡-diolato-(or£)-N(2disulfamoyl-3,34-amino-2-hydroxy)phenylazo]5¢)-g-6,6¢-bis[2-amino-4-(or6)-hydroxy-¡)-N(2):N¡,5£-2,1¢-disulfonatobis(naphthalene-chromate

¡azobenzene-1,2¡-diolato-O(1),O(2¡))-8.6.2000ENOfficialJournaloftheEuropeanCommunitiesL136/27IndexNoChemicalname

substances

relatedNotestoECNoCASNoClassificationLabellingConcentrationlimits

relatedNotesprepara-totions

611-044-00󰂖0

Abis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naph-mixtureof:tert-alkyl(C12-C14)ammonium403-720-7117527-94-3N;R51-53

N

thalenolato(2-)]-chromate(1-);R:(C12-C14)ammoniumtert-alkyl

S:61

51/53trophenyl)azo]-2-naphthalenolato(2-)]-chro-bis[1-[(2-hydroxy-4-ni-mate(1-);bis[1-[[5-(1,1-dimethylpropyl)-2-hydroxy-3-ni-tert-alkyl(C12-C14)ammonium

trophenyl]azo]-2-naphthalenolato(2-)]-chro-mate(1-);[[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naph-tert-alkyl(C12-C14)ammonium-thalenolato(2-)]-[1-[(2-hydroxy-5-nitrophe-nyl)azo]-2-naphthalenolato(2-)]]-chromate(1-);tert-alkyl(C12-C14)ammonium-[[1-[[5-(1,1-di-methylpropyl)-2-hydroxy-3-nitrophenyl]azo]-2-naphthalenolato(2-)]-[1-[(2-hydroxy-5-nitro-phenyl)azo]-2-naphthalenolato(2-)]]-chromate(1-);5)-nitro-2-oxidophenylazo)-2-naphtholato)tert-alkyl(C12-C14)ammonium((1-(4(or(1-(3-nitro-2-oxido-5-pentylphenylazo)-2-naph-tholato))chromate(1-)

611-045-00-6

2-[4-[N-(4-acetoxybutyl)-N-ethyl]amino-2-methylphenylazo]-3-acetyl-5-nitrothiophene

404-830-8󰂗R53

R:S:61

53611-046-00-14,4¡-diamino-2-methylazobenzene407-590-243151-99-1

T;Xn;R25T;R43

R48/22R:N

N;R50-53

S:(1/2-)22-28-36/37-45-60-61

25-43-48/22-50/53

611-047-00-7

A2-[[4-[N-ethyl-N-(2-acetoxyethyl)amino]phe-1:1mixtureof:

407-0-3111381-11-4R53

R:nyl]azo]-5,6-dichlorobenzothiazole;2-S:61

53[[4-[N-ethyl-N-(2-acetoxyethyl)amino]phe-nyl]azo]-6,7-dichlorobenzothiazole

611-048-00-2

A2-[[4-[bis(2-acetoxyethyl)amino]phenyl]azo]-(1:1)mixtureof:

407-900-6111381-12-5R53

R:5,6-dichlorobenzothiazole;S:61

53ethyl)amino]phenyl]azo]-6,7-dichlorobenzothia-2-[[4-[bis(2-acetoxy-zole

L136/28ENOfficialJournaloftheEuropeanCommunities8.6.2000IndexNoChemicalname

substances

relatedNotestoECNoCASNoClassificationLabellingConcentrationlimits

relatedNotesprepara-totions

611-049-00-8

7-[4-(3-diethylaminopropylamino)-6-(3-diethy-lammoniopropylamino)-1,3,5-triazin-408-000-6118658-98-3

Xn;2-ylamino]-4-hydroxy-3-(4-phenylazopheny-R43R48/22Xn

lazo)-naphthalene-2-sulfonate,R52-53R:S:(2-(22-36/37-61

43-48/22-52/53acid(2:1:1)

aceticacid,lactic611-051-00-92-(4-(N-ethyl-N-(2-hydroxy)ethyl)amino-2-methylphenyl)azo-6-methoxy-3-methylbenzo-411-110-7136213-74-6N;R50-53

N

thiazoliumchloride

R:S:60-6150/53611-052-00-4monosodium[(2-hydroxy-3,5-dinitrophenyl)azo]phe-aqua-[5-[[2,4-dihydroxy-5-400-720-9󰂗R52-53

R:nyl]azo]-2-naphthalensulfonate],ironcomplexS:61

52/53612-156-00-2Achloride;mixtureof:trihexadecylmethylammonium405-620-9󰂗

Xi;loride

dihexadecyldimethylammoniumch-N;R50-53

R41Xi;R:N

S:(2-)26-39-60-6141-50/53

612-157-00-8(Z)-1-benzo[b]thien-2-ylethanonehydrochloride

oxime410-780-8󰂗

Xn;Xi;Xn;R43

R41R22-48/22R:22-41-43-48/22-51/53N

N;R51-53S:(2-)22-26-36/37/39-61

612-158-00-3

Abis(5-dodecyl-2-hydroxybenzaldoximate)mixtureof:

410-820-4󰂗

R53

R:(II)S:61

534-dodecylsalicylaldoxime

C12-alkylgroupisbranched;copper612-159-00-9Reactiondiamineproductsof:trimethylhexamethylene410-880-1󰂗

Xn;2,2,4-trimethyl-1,6-hexanediamine(amixtureof

C;R34R22C;2,4,4-trimethyl-1,6-hexanediamine,andN;R50-53

R:N

S:(1/2-)23-26-36/37/39-45-22-34-50/53

listed),60-61

(mono[(C10-C16-alkyloxy)methyl]oxiraneEpoxide8

EINECSderivatives)andp-toluene-sulfonicacid613-149-00-72-tert-butyl-5-(4-tert-butylbenzylthio)-4-chloro-pyridazin-3(2H)-one

405-700-39-71-3

T;N;R23/25R50-53

T;R:N

S:(1/2-)36/37-45-60-6123/25󰂖50/53

613-150-00-2

2,2(1H-benzimidazo[2,1-b]benzo[l,m,n][3,8]phen-¡-[3,3¡-(piperazine-1,4-diyl)dipropyl]bis406-295-6󰂗R53

R:53anthroline-1,3,6-trione

S:61

8.6.2000ENOfficialJournaloftheEuropeanCommunitiesL136/29IndexNoChemicalname

Notessubstances

relatedtoECNoCASNoClassificationLabellingConcentrationlimits

relatedNotesprepara-totions

613-151-00-8

1-(3-mesyloxy-5-trityloxymethyl-2-D-threofu-ryl)thymine

406-360-9104218-44-2R53

R:S:61

53613-152-00-3phenylmate

N-(4,6-dimethoxypyrimidin-2-yl)carba-406-600-2392-03-0

R43

N;R51-53

Xi;R:N

S:(2-)24-37-6143-51/53613-153-00-92,3,5-trichloropyridine407-270-216063-70-0R52-53

R:S:6152/53613-154-00-42-amino-4-chloro-6-methoxypyrimidine410-050-95734--5Xn;R22

XnR:S:(2-)2222613-155-00-X5-chloro-2,3-difluoropyridine410-090-7402-43-7

R10Xn;Xn

R52-53R22R:S:(2-)23-36-6110-22-52/53613-156-00-52-butyl-4-chloro-5-formylimidazole410-260-083857-96-9

R43

N;R51-53

Xi;R:N

S:(2-)24-37-6143-51/53613-157-00-02,4-diamino-5-methoxymethylpyrimidine410-330-054236-98-5

Xn;Xi;R36

R22-48/22Xn

R:S:(2-)22-26-3622-36-48/22613-158-00-62,3-dichloro-5-trifluoromethyl-pyridine410-340-569045-84-7

Xn;Xi;R20/22Xn;R43

R41R:N

N;R51-53S:(2-)24-26-37/39-61

20/22-41-43-51/53613-159-00-1

4-[2-[4-(1,1-dimethylethyl)phenyl]-ethoxy]qui-nazoline

410-580-0120928-09-8

T;Xn;R25R20T;R:N

N;R50-53S:(1/2-)37-45-60-6120-25-50/53

613-160-00-7

(1S)-2-methyl-2,5-diazobicyclo[2.2.1]heptanedihydrobromide

411-000-9125224-62-6

R43

XiR:S:(2-)24-37

43

615-022-00-1

methylboxylate

3-isocyanatosulfonyl-2-thiophene-car-410-550-779277-18-2

E;R14

R2E;Xn

Xn;R:R42/43

R48/22S:(2-)22-30-35-36/37

2-14-42/43-48/22L136/30ENOfficialJournaloftheEuropeanCommunities8.6.2000IndexNoChemicalname

relatedNotessubstances

relatedNotestoECNoCASNoClassificationLabellingConcentrationlimits

prepara-totions

615-023-00-7

2-(isocyanatosulfonylmethyl)benzoicmethylester;(alt.):methyl

acid410-900-983056-32-0

R10Xn

2-(isocyanatosulfonylmethyl)benzoate

R14

Muta.R:Xn;Cat.3;S:(2-)23-26-36/37/39

10-14-20-40-41-42-48/22Xi;R20-48/22R40R42R41616-044-00-4

N-(3,5-dichloro-4-ethyl-2-hydroxyphenyl)-2-(3-pentadecylphenoxy)-butanamide402-510-2󰂗

N;R51-53

N

R:S:61

51/53616-045-00-X25¡¡-(4-chloro-3-cyano-5-formyl-2-thienylazo)--diethylamino-2-methoxyacetanilide405-190-2122371-93-1

R43R53Xi

R:S:(2-)22-24-37-6143-53

616-046-00-5N-(2-(6-chloro-7-methylpyrazolo(1,5-b)-1,2,4-triazol-4-yl)propyl)-2-(2,4-di-tert-pentylp-406-390-2󰂗

N;R50-53

N

henoxy)octanamide

R:S:60-6150/53616-047-00-0A2,2mixture406-0-0󰂗R43

Xialkylacetamide;¡,2¢,2£-(ethylenedinitrilotetrakis-N,N-di(C16)of:

R:ethylenedinitrilotetrakis-N,N-di(C18)alkylaceta-2,2¡,2¢,2£-S:(2-)24-37

43

mide

616-048-00-63¡-trifluoromethylisobutyranilide

406-740-41939-27-1

Xn;N;R51-53R48/22Xn;R:N

S:(2-)22-36-6148/22-51/53616-049-00-1

2-(2,4-bis(1,1-dimethylethyl)phenoxy)-N-(3,5-dichloro-4-ethyl-2-hydroxyphenyl)-hex-408-150-299141--6

R53

R:anamide

S:61

53616-050-00-7N-[2,5-dichloro-4-(1,1,2,3,3,3-hexafluoropro-poxy)-phenyl-aminocarbonyl]-2,6-difluoroben-410-690-9103055-07-8

R43

N;R50-53Xi;zamide

R:N

S:(2-)24-37-60-6143-50/53

616-051-00-2A-bis(Nmixtureof:2,4

411-070-0󰂗

R53

R:-bis(N¡¡-(4-methylphenyl)-ureido)-toluene;-(4-methylphenyl)-ureido)-toluene2,6S:61

53617-015-00-9bis(4-methylbenzoyl)peroxide

407-950-95-85-2

E;O;R2E;N;R7R50-53R:N

S:(2-)7-14-36/37/39-47-60-612-7-50/53

650-032-00-X

cyproconazole(2RS,3RS;2RS,3(ISO)

󰂗94361-06-5

Repr.Xn;cyclopropyl-1-(1SRXn;Cat.3;R63R:yl)butan-2-ol

H)-2-(4-chlorophenyl)-3--1,2,4-triazol-1-N;R50-53

R22S:(2-)36/37-60-61

22-50/53-63N

8.6.2000ENOfficialJournaloftheEuropeanCommunitiesL136/31L136/32ENOfficialJournaloftheEuropeanCommunities8.6.2000

ANNEX2

R66

IT:L'esposizioneripetutapuòprovocaresecchezzaescrepolaturedellapelle.(DoesnotconcerntheESversion)(DoesnotconcerntheDAversion)(DoesnotconcerntheDEversion)(DoesnotconcerntheELversion)(DoesnotconcerntheENversion)(DoesnotconcerntheFRversion)(DoesnotconcerntheNLversion)(DoesnotconcernthePTversion)(DoesnotconcerntheFIversion)(DoesnotconcerntheSVversion)

8.6.2000ENOfficialJournaloftheEuropeanCommunitiesL136/33

ANNEX3A

S23FR:

Nepasrespirerlesgaz/fumées/vapeurs/aérosols[terme(s)approprié(s)àindiquerparlefabricant].

(DoesnotconcerntheESversion)(DoesnotconcerntheDAversion)(DoesnotconcerntheDEversion)(DoesnotconcerntheELversion)(DoesnotconcerntheENversion)(DoesnotconcerntheITversion)(DoesnotconcerntheNLversion)(DoesnotconcernthePTversion)(DoesnotconcerntheFIversion)(DoesnotconcerntheSVversion)S26

DE:BeiBerührungmitdenAugensofortgründlichmitWasserabspülenundArztkonsultieren.(DoesnotconcerntheESversion)(DoesnotconcerntheDAversion)(DoesnotconcerntheELversion)(DoesnotconcerntheENversion)(DoesnotconcerntheFRversion)(DoesnotconcerntheITversion)(DoesnotconcerntheNLversion)(DoesnotconcernthePTversion)(DoesnotconcerntheFIversion)(DoesnotconcerntheSVversion)S56

DE:DiesenStoffundseinenBehälterderProblemabfallentsorgungzuführen.

EN:Disposeofthismaterialanditscontainertohazardousorspecialwastecollectionpoint.IT:

Smaltirequestomaterialeeirelativicontenitoriinunpuntodiraccoltadirifiutipericolosiospeciali.

(DoesnotconcerntheESversion)(DoesnotconcerntheDAversion)(DoesnotconcerntheELversion)(DoesnotconcerntheFRversion)(DoesnotconcerntheNLversion)(DoesnotconcernthePTversion)(DoesnotconcerntheFIversion)(DoesnotconcerntheSVversion)

L136/34ENOfficialJournaloftheEuropeanCommunities8.6.2000

ANNEX3B

S27/28

DE:BeiBerührungmitderHautbeschmutzte,getränkteKleidungsofortausziehenundHautsofortmitviel󰂅

abwaschen(vomHerstelleranzugeben).(DoesnotconcerntheESversion)(DoesnotconcerntheDAversion)(DoesnotconcerntheELversion)(DoesnotconcerntheENversion)(DoesnotconcerntheFRversion)(DoesnotconcerntheITversion)(DoesnotconcerntheNLversion)(DoesnotconcernthePTversion)(DoesnotconcerntheFIversion)(DoesnotconcerntheSVversion)S29/56ES:

Notirarlosresiduosporeldesagüe;elimíneseestasustanciaysurecipienteenunpuntoderecogidapúblicaderesiduosespecialesopeligrosos.

DE:NichtindieKanalisationgelangenlassen;diesenStoffundseinenBehälterderProblemabfallentsorgungzuführen.EN:Donotemptyintodrains,disposeofthismaterialanditscontainertohazardousorspecialwastecollection

point.IT:

Nongettareiresiduinellefognature;smaltirequestomaterialeeirelativicontenitoriinunpuntodiraccoltadirifiutipericolosiospeciali.

NL:Afvalnietindegootsteenwerpen;dezestofendeverpakkingnaareeninzamelpuntvoorgevaarlijkofbijzonder

afvalbrengen.SV:Tömejiavloppet,lämnadettamaterialochdessbehållaretillinsamlingsställeförfarligtavfall.(DoesnotconcerntheDAversion)(DoesnotconcerntheELversion)(DoesnotconcerntheFRversion)(DoesnotconcernthePTversion)(DoesnotconcerntheFIversion)

8.6.2000ENOfficialJournaloftheEuropeanCommunitiesL136/35

ANNEX4A

󰂑B.10.MUTAGENICITY󰂗INVITROMAMMALIANCHROMOSOMEABERRATIONTEST

1.METHOD

ThismethodisareplicateoftheOECDTG473,InVitroMammalianChromosomeAberrationTest(1997).

1.1.INTRODUCTION

Thepurposeoftheinvitrochromosomalaberrationtestistoidentifyagentsthatcausestructuralchromosomeaberrationsinculturedmammaliancells(1)(2)(3).Structuralaberrationsmaybeoftwotypes,chromomeorchromatid.Withthemajorityofchemicalmutagens,inducedaberrationsareofthechromatidtype,butchromosome-typeaberrationsalsooccur.Anincreaseinpolyploidymayindicatethatachemicalhasthepotentialtoinducenumericalaberrations.However,thismethodisnotdesignedtomeasurenumericalaberrationsandisnotroutinelyusedforthatpurpose.Chromosomemutationsandrelatedeventsarethecauseofmanyhumangeneticdiseasesandthereissubstantialevidencethatchromosomemutationsandrelatedeventscausingalterationsinoncogenesandtumoursuppressorgenesofsomaticcellsareinvolvedincancerinductioninhumansandexperimentalanimals.

Theinvitrochromosomeaberrationtestmayemployculturesofestablishedcellslines,cellstrainsorprimarycellcultures.Thecellsusedareselectedonthebasisofgrowthabilityinculture,stabilityofthekaryotype,chromosomenumber,chromosomediversityandspontaneousfrequencyofchromosomeaberrations.Testsconductedinvitrogenerallyrequiretheuseofanexogenoussourceofmetabolicactivation.Thismetabolicactivationsystemcannotmimicentirelythemammalianinvivoconditions.CareshouldbetakentoavoidconditionswhichwouldleadtopositiveresultswhichdonotreflectintrinsicmutagenicityandmayarisefromchangesinpH,osmolalityorhighlevelsofcytotoxicity(4)(5).

Thistestisusedtoscreenforpossiblemammalianmutagensandcarcinogens.Manycompoundsthatarepositiveinthistestaremammaliancarcinogens;however,thereisnotaperfectcorrelationbetweenthistestandcarcinogenicity.CorrelationisdependentonchemicalclassandthereisincreasingevidencethattherearecarcinogensthatarenotdetectedbythistestbecausetheyappeartoactthroughmechanismsotherthandirectDNAdamage.

SeealsoGeneralIntroductionPartB.

1.2.DEFINITIONS

Chromatid-typeaberration:structuralchromosomedamageexpressedasbreakageofsinglechromatidsorbreakageandreunionbetweenchromatids.

Chromatid-typeaberration:structuralchromosomedamageexpressedasbreakage,orbreakageandreunion,ofbothchromatidsatanidenticalsite.

Endoreduplication:aprocessinwhichafteranSperiodofDNAreplication,thenucleusdoesnotgointomitosisbutstartsanotherSperiod.Theresultischromosomeswith4,8,16,󰂅chromatids.

Gap:anachromaticlesionsmallerthanthewidthofonechromatid,andwithminimummisalignmentofthecromatids.

Mitoticindex:theratioofcellsinmetaphasedividedbythetotalnumberofcellsobservedinapopulationofcells;anindicationofthedegreeofproliferationofthatpopulation.

Numericalaberration:achangeinthenumerofchromosomesfromthenormalnumbercharacteristicofthecellsutilised.

L136/36ENOfficialJournaloftheEuropeanCommunities8.6.2000

Polyploidy:amultipleofthehaploidchromosomenumber(n)otherthanthediploidnumber(i.e.3n,4n,andsoon).

Structuralaberration:achangeinchromosomestructuredetectablebymicroscopicexaminationofthemetaphasestageofcelldivision,observedasdeletionsandfragments,intrachangesorinterchanges.

1.3.PRINCIPLEOFTHETESTMETHOD

Cellculturesareexposedtothetestsubstancebothwithandwithoutmetabolicactivation.Atpredeterminedintervalsafterexposureofcellculturestothetestsubstance,theyaretreatedwithametaphasearrestingsubstance(e.g.Colcemid®orcolchicine),harvested,stainedandmetaphasecellsareanalysedmicroscopicallyforthepresenceofchromosomeaberrations.

1.4.DESCRIPTIONOFTHETESTMETHOD

1.4.1.Preparations

1.4.1.1.Cells

Avarietyofcelllines,strainsorprimarycellcultures,includinghumancells,maybeused(e.g.Chinesehamsterfibroblasts,humanorothermammalianperipheralbloodlymphocytes).

1.4.1.2.Mediaandcultureconditions

Appropriateculturemedia,andincubationconditions(culturevessels,CO2concentration,temperatureandhumidity)shouldbeusedinmaintainingcultures,Establishedcelllinesandstrainsshouldbecheckedroutinelyforstabilityinthemodalchromosomenumberandtheabsenceofmycoplasmacontaminationandshouldnotbeusedifcontaminated.Thenormalcellcycletimeforthecellsandcultureconditionsusedshouldbeknown.

1.4.1.3.Preparationofcultures

Establishedcelllinesandstrains:cellsarepropagatedfromstockcultures,seededinculturemediumatadensitysuchthatthecultureswillnotreachconfluencybeforethetimeofharvest,andincubatedat37°C.Lymphocytes:wholebloodtreatedwithananti-coagulant(e.g.heparin)orseparatedlymphocytesobtainedfromhealthysubjectsareaddedtotheculturemediumcontainingamitogen(e.g.phytohaemagglutinin)andincubatedat37°C.

1.4.1.4.Metabolicactivation

Cellsshouldbeexposedtothetestsubstancebothinthepresenceandabsenceofanappropriatemetabolicactivationsystem.Themostcommonlyusedsystemisacofactor-supplementedpost-mitochondrialfraction(S9)preparedfromtheliversofrodentstreatedwithenzyme-inducingagentssuchasAroclor1254(6)(7)(8)(9),oramixtureofphenobarbitoneandb-naphthoflavone(10)(11)(12).

Thepost-mitochondrialfractionisusuallyusedatconcentrationsintherangefrom1󰂗10%v/vinthefinaltestmedium.Theconditionofametabolicactivationsystemmaydependupontheclassofchemicalbeingtested.Insomecasesitmaybeappropriatetoutilisemorethanoneconcentrationofpost-mitochondrialfraction.

Anumberofdevelopments,includingtheconstructionofgeneticallyengineeredcelllinesexpressingspecificactivatingenzymes,mayprovidethepotentialforendogenousactivation.Thechoiceofthecelllinesusedshouldbescientificallyjustified(e.g.bytherelevanceofthecytochromeP450isoenzymeforthemetabolismofthetestsubstance).

8.6.2000

1.4.1.5.

ENTestsubstance/Preparation

OfficialJournaloftheEuropeanCommunitiesL136/37

Solidtestsubstancesshouldbedissolvedorsuspendedinappropriatesolventsorvehiclesanddilutedifappropriatepriortotreatmentofthecells.Liquidtestsubstancesmaybeaddeddirectlytothetestsystemsand/ordilutedpriortotreatment.Freshpreparationsofthetestsubstanceshouldbeemployedunlessstabilitydatademonstratetheacceptabilityofstorage.

1.4.2.Testconditions

1.4.2.1.Solvent/vehicle

Thesolvent/vehicleshouldnotbesuspectedofchemicalreactionwiththetestsubstanceandshouldbecompatiblewiththesurvivalofthecellsandtheS9activity.Ifotherthanwell-knownsolvent/vehiclesareused,theirinclusionshouldbesupportedbydataindicatingtheircompatibility.Itisrecommendedthatwhereverpossible,theuseofanaqueoussolvent/vehiclebeconsideredfirst.Whentestingwater-unstablesubstances,theorganicsolventsusedshouldbefreeofwater.Watercanberemovedbyaddingamolecularsieve.

1.4.2.2.Exposureconcentrations

Amongthecriteriatobeconsideredwhendeterminingthehighestconcentrationarecytotoxicity,solubilityinthetestsystemandchangesinpHorosmolality.

Cytotoxicityshouldbedeterminedwithandwithoutmetabolicactivationinthemainexperimentusinganappropriateindicationofcellintegrityandgrowth,suchasdegreeofconfluency,viablecellcounts,ormitoticindex.Itmaybeusefultodeterminecytotoxicityandsolubilityinapreliminaryexperiment.

Atleastthreeanalysableconcentrationsshouldbeused.Wherecytotoxicityoccurs,theseconcentrationsshouldcoverarangefromthemaximumtolittleornotoxicity;thiswillusuallymeantheconcentrationsshouldbeseparatedbynomorethanafactorbetween2andÓ10.Atthetimeofharvesting,thehighestconcentrationshouldshowasignificantreductionindegreeofconfluency,cellcountormitoticindex(allgreaterthan50%).Themitoticindexisonlyanindirectmeasureofcytotoxic/cytostaticeffectsanddependsonthetimeaftertreatment.However,themitoticindexisacceptableforsuspensionculturesinwhichothertoxicitymeasurementsmaybecumbersomeandimpractical.Informationoncellcyclekinetics,suchasaveragegenerationtime(AGT),couldbeusedassupplementaryinformationAGT,however,isanoverallaveragethatdoesnotalwaysrevealtheexistenceofdelayedsubpopulations,andevenslightincreasesinaveragegenerationtimecanbeassociatedwithverysubstantialdelayinthetimeofoptimalyieldofaberrations.

Forrelativelynon-cytotoxicsubstances,themaximumtestconcentrationshouldbe5µl/ml,5mg/mlor0,01M,whicheveristhelowest.

Forrelativelyinsolublesubstancesthatarenottoxicatconcentrationslowerthantheinsolubleconcentration,thehighestdoseusedshouldbeaconcentrationabovethelimitofsolubilityinthefinalculturemediumattheendofthetreatmentperiod.Insomecases(e.g.whentoxicityoccursonlyathigherthanthelowestinsolubleconcentration),itisadvisabletotestatmorethanoneconcentrationwithvisibleprecipitation.Itmaybeusefultoassesssolubilityatthebeginningandtheendofthetreatment,assolubilitycanchangeduringthecourseofexposureinthetestsystemduetopresenceofcells,S9serum,etc.Insolubilitycanbedetectedbyusingtheunaidedeye.Theprecipitateshouldnotinterferewiththescoring.

1.4.2.3.Negativeandpositivecontrols

Concurrentpositiveandnegative(solventorvehicle)controls,bothwithandwithoutmetabolicactivation,shouldbeincludedineachexperiment.Whenmetabolicactivationisused,thepositivecontrolchemicalshouldbetheonethatrequiresactivationtogiveamutagenicresponse.

Positivecontrolsshouldemployaknownelastogenatexposurelevelsexpectedtogiveareproducibleanddetectableincreaseoverbackgroundwhichdemonstratesthesensitivityofthetestsystem.

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Positivecontrolconcentrationsshouldbechosensothattheeffectsareclearbutdonotimmediatelyrevealtheidentityofthecodedslidestothereader.Examplesofpositivecontrolsubstancesinclude:

MetabolicactivationconditionSubstanceCASNoEinecsNo

Absenceofexogenousmetabolicactivation

methylmethanesulphonateethylmethanesulphonateethylnitrosoureamitomycinC

4-nitroquinoline-N-oxide

66-27-362-50-0759-73-950-07-756-57-550-32-850-18-06055-19-2

200-625-0200-536-7212-072-2200-008-6200-281-1200-028-5200-015-4

Presenceofexogenousmetabolicactivation

benzo[a]pyrenecyclophosphamide

cyclophosphamidemonohydrate

Otherappropriatepositivecontrolsubstancesmaybeused.Theuseofchemicalclass-relatedpositivecontrolchemicalsshouldbeconsidered,whenavailable.

Negativecontrols,consistingofsolventorvehiclealoneinthetreatmentmedium,andtreatedinthesamewayasthetreatmentcultures,shouldbeincludedforeveryharvesttime.Inaddition,untreatedcontrolsshouldalsobeusedunlesstherearehistoricalcontroldatademonstratingthatnodeleteriousormutageniceffectsareinducedbythechosensolvent.

1.4.3.Procedure

1.4.3.1.Treatmentwiththetestsubstance

Proliferatingcellsaretreatedwiththetestsubstanceinthepresenceandabsenceofametabolicactivationsystem.Treatmentoflymphocytesshouldcommenceatabout48hoursaftermitogenicstimulation.

1.4.3.2.

Duplicateculturesshouldnormallybeusedateachconcentration,andarestronglyrecommendedfornegative/solventcontrolcultures.Whereminimalvariationbetweenduplicateculturescanbedemonstrated(13)(14),fromhistoricaldata,itmaybeacceptableforsingleculturestobeusedateachconcentration.Gaseousorvolatilesubstancesshouldbetestedbyappropriatemethods,suchasinsealedculturevessels(15)(16).

1.4.3.3.Cultureharvesttime

Inthefirstexperiment,cellsshouldbeexposedtothetestsubstance,bothwithandwithoutmetabolicactivation,for3󰂗6hours,andsampledatatimeequivalenttoabout1,5normalcellcyclelengthafterthebeginningoftreatment(12).Ifthisprotocolgivesnegativeresultsbothwithandwithoutactivation,anadditionalexperimentwithoutactivationshouldbedone,withcontinuoustreatmentuntilsamplingatatimeequivalenttoabout1,5normalcellcyclelengths.Certainchemicalsmaybemorereadilydetectedbytreatment/samplingtimeslongerthan1,5cyclelengths.Negativeresultswithmetabolicactivationneedtobeconformedonacase-by-casebasis.Inthosecaseswhereconfirmationofnegativeresultsisnotconsiderednecessary,justificationshouldbeprovided.

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1.4.3.4.

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CellculturesaretreatedwithColcemid®orcolchicineusuallyfor1󰂗3hourspriortoharvesting.Eachcellcultureisharvestedandprocessedseparatelyforthepreparationofchromosomes.Chromosomepreparationinvolveshypotonictreatmentofthecells,fixationandstaining.

1.4.3.5.Analysis

Allslides,includingthoseofpositiveandnegativecontrols,shouldbeindependentlycodedbeforemicroscopicanalysis.Sincefixationproceduresoftenresultinthebreakageofaproportionofmetaphasecellswithlossofchromosomes,thecellsscoredshouldthereforecontainanumberofcentromeresequaltothemodalnumberÊ2forallcelltypes.Atleast200well-spreadmetaphasesshouldbescoredperconcentrationandcontrol,equallydividedamongsttheduplicates,ifapplicable.Thisnumbercanbereducedwhenahighnumberofaberrationsisobserved.

Thoughthepurposeofthetestistodetectstructuralchromosomeaberrations,itisimportanttorecordpolyploidyandendoreduplicationwhentheseeventsareseen.

2.DATA

2.1.TREATMENTOFRESULTS

Theexperimentalunitisthecell,andthereforethepercentageofcellswithstructuralchromosomeaberration(s)shouldbeevaluated.Differenttypesofstructuralchromosomeaberrationsshouldbelistedwiththeirnumbersandfrequenciesforexperimentalandcontrolcultures.Gapsarerecordedseparatelyandreportedbutgenerallynotincludedinthetotalaberrationfrequency.

Concurrentmeasuresofcytotoxicityforalltreatedandnegativecontrolculturesinthemainaberrationexperimentsshouldalsoberecorded.

Individualculturedatashouldbeprovided.Additionally,alldatashouldbesummarisedintabularform.Thereisnorequirementforverificationofaclearpositiveresponse.Equivocalresultsshouldbeclarifiedbyfurthertestingpreferablyusingmodificationofexperimentalconditions.Theneedtoconfirmnegativeresultshasbeendiscussedin1.4.3.3.Modificationofstudyparameterstoextendtherangeofconditionsassessedshouldbeconsideredinfollow-upexperiments.Studyparametersthatmightbemodifiedincludetheconcentrationspacingandthemetabolicactivationconditions.

2.2.EVALUATIONANDINTERPRETATIONOFRESULTS

Thereareseveralcriteriafordeterminingapositiveresult,suchasaconcentration-relatedincreaseorareproducibleincreaseinthenumberofcellswithchromosomeaberrations.Biologicalrelevanceoftheresultsshouldbeconsideredfirst.Statisticalmethodsmaybeusedasanaidinevaluatingthetestresults(3)(13).Statisticalsignificanceshouldnotbetheonlydeterminingfactorforapositiveresponse.

Anincreaseinthenumberofpolyploidcellsmayindicatethatthetestsubstancehasthepotentialtoinhibitmitoticprocessesandtoinducenumericalchromosomeaberrations.Anincreaseinthenumberofcellswithendoreduplicatedchromosomesmayindicatethatthetestsubstancehasthepotentialtoinhibitcellcycleprogression(17)(18).

Atestsubstanceforwhichtheresultsdonotmeettheabovecriteriaisconsiderednon-mutagenicinthissystem.

Althoughmostexperimentswillgiveclearlypositiveornegativeresults,inrarecasesthedatasetwillprecludemakingadefinitejudgementabouttheactivityofthetestsubstance.Resultsmayremainequivocalorquestionableregardlessofthenumberoftimestheexperimentisrepeated.

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Positiveresultsfromtheinvitrochromosomeaberrationtestindicatethatthetestsubstanceinducesstructuralchromosomeaberrationsinculturedmammaliansomaticcells.Negativeresultsindicatethat,underthetestconditions,thetestsubstancedoesnotinducechromosomeaberrationsinculturedmammaliansomaticcells.

3.REPORTINGTESTREPORT

Thetestreportmustincludethefollowinginformation:Solvent/vehicle:

󰂗justificationforchoiceofvehicle,

󰂗solubilityandstabilityofthetestsubstanceinsolvent/vehicle,ifknown.Cells:

󰂗typeandsourceofcells,

󰂗karyotypefeaturesandsuitabilityofthecelltypeused,󰂗absenceofmycoplasma,ifapplicable,󰂗informationoncellcyclelength,

󰂗sexofblooddonors,wholebloodorseparatedlymphocytes,mitogenused,󰂗numberofpassages,ifapplicable,

󰂗methodsformaintenanceofcellculture,ifapplicable,󰂗modalnumberofchromosomes.Testconditions:

󰂗identityofmetaphasearrestingsubstance,itsconcentrationanddurationofcellexposure,

󰂗rationaleforselectionofconcentrationsandnumberofculturesincluding,e.g.cytotoxicitydataand

solubilitylimitations,ifavailable,󰂗compositionofmedia,CO2concentrationifapplicable,󰂗concentrationoftestsubstance,

󰂗volumeofvehicleandtestsubstanceadded,󰂗incubationtemperature,󰂗incubationtime,󰂗durationoftreatment,

󰂗celldensityatseeding,ifappropriate,

󰂗typeandcompositionofmetabolicactivationsystem,includingacceptabilitycriteria,󰂗positiveandnegativecontrols,󰂗methodsofslidepreparation,󰂗criteriaforscoringaberrations,

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󰂗numberofmetaphasesanalysed,

󰂗methodsforthemeasurementsoftoxicity,

󰂗criteriaforconsideringstudiesaspositive,negativeorequivocal,Results:

󰂗signsoftoxicity,e.g.degreeofconfluency,cellcycledata,cellcounts,mitoticindex,󰂗signsofprecipitation,

󰂗dataonpHandosmolalityofthetreatmentmedium,ifdetermined,󰂗definitionforaberrations,includinggaps,

󰂗numberofcellswithchromosomeaberrationsandtypeofchromosomeaberrationsgivenseparatelyfor

eachtreatedandcontrolculture,󰂗changesinploidyifseen,

󰂗dose-responserelationship,wherepossible,󰂗statisticalanalyses,ifany,

󰂗concurrentnegative(solvent/vehicle)andpositivecontroldata,

󰂗historicalnegative(solvent/vehicle)andpositivecontroldata,withranges,meansandstandarddeviations.Discussionofresults.Conclusions.

4.REFERENCES

(1)Evans,H.J.(1976),CytologicalMethodsforDetectingChemicalMutagens,in:Chemicalmutagens,

PrinciplesandMethodsfortheirDetection,Vol.4,Hollaender,A.(ed)PlenumPress,NewYorkandLondon,pp.1󰂗29.(2)Ishidate,M.Jr.andSofumi,T.(1985),TheInVitroChromosomalAberrationTestUsingChinese

HamsterLung(CHL)FibroblastCellsinCulture,in:ProgressinMutationResearch,Vol.5,Ashby,J.etal.,(eds)ElsevierSciencePublishers,Amsterdam-NewYork-Oxford,pp.427󰂗432.(3)Galloway,S.M.,Armstrong,M.J.,Reuben,C.,Colman,S.,Brown,B.,Cannon,C.,Bloom,A.D.,

Nakamura,F.,Ahmed,M.,Duk,S.,Rimpo,J.,Margolin,G.H.,Resnick,M.A.,Anderson,G.andZeigerE.(1978),ChromosomeaberrationandsisterchromaticexchangesinChinesehamsterovarycells:Evaluationof108chemicals,Environs,Molec.Mutagen10(suppl.10),pp.1󰂗175.(4)Scott,D.,Galloway,S.M.,Marshall,R.R.,Ishidate,M.Jr.,Brusick,D.,Ashby,J.andMyhr,B.C.(1991),

GenotoxicityunderExtremeCultureConditions.AreportfromICPEMCTaskGroup9,MutationRes.,257,pp.147󰂗204.(5)Morita,T.,Nagaki,T.,Fukuda,I.andOkumura,K.,(1992),ClastogenicityoflowpHtoVarious

CulturedMammalianCells,MutationRes.,268,pp.297󰂗305.(6)Ames,B.N.,McCann,J.andYamasaki,E.(1975),MethodsforDetectingCarcinogensandMutagens

withtheSalmonella/MammalianMicrosomeMutagenicityTest,MutationRes.,31,pp.347󰂗3.(7)Maron,D.M.andAmes,B.N.(1983),RevisedMethodsfortheSalmonellaMutagenicityTest,Mutation

Res.,113,pp.173󰂗215.

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(8)Natarajan,A.T.,Tates,A.D.,vanBuul,P.P.W.,Meijers,M.anddeVogel,N.(1976),Cytogenetic

EffectsofMutagen/CarcinogensafterActivationinaMircrosomalSystemInVitro,I.InductionofChromosomeAberrationsandSisterChromatidExchangebyDiethylnitrosamine(DEN)andDimethylnitrosamine(DMN)inCHOCellsinthePresenceofRat-LiverMicrosomes,MutationRes.,37,pp.83󰂗90.(9)Matsuoka,A.,Hayashi,M.andIshidate,M.Jr.(1979),ChromosomalAberrationTestson29Chemicals

CombinedwithS9MixInVitro,MutationRes.,66,pp.277󰂗290.(10)Elliot,B.M.,Combes,R.D.,Elcombe,C.R.,Gatehouse,D.G.,Gibson,G.G.,Mackay,J.M.andWolf,

R.C.(1992),ReportofUKEnvironmentalMutagenSocietyWorkingParty.AlternativetoAroclor1254-inducedS9inInVitroGenotoxicityAssays,Mutagenesis,7,pp.175󰂗177.(11)Matsushima,T.,Sawamura,M.,Hara,K.andSugimura,T.(1976),A.SafeSubstituteforPolychlorinated

BiphenylsasanInducerofMetabolicActivationSystems,in:deSerres,F.J.Fouts,J.R.Bend,J.R.andPhilpot,R.M.(eds),InVitroMetabolicActivationinMutagenesisTesting,Elsevier,North-Holland,pp.85󰂗88.(12)Galloway,S.M.,Aardema,M.J.,Ishidate,M.Jr.,Ivett,J.L.,Kirkland,D.J.Morita,T.,Mosesso,P.,

Sofumi,T.(1994),ReportfromWorkingGrouponinInVitroTestsforChromosomalAberrations,MutationRes.,312,pp.241󰂗261.(13)Richardson,C.,Williams,D.A.,Allen,J.A.,Amphlett,G.,Chanter,D.O.andPhillips,B.(19),

AnalysisofDatafromInVitroCytogeneticAssays,in:StatisticalEvaluationofMutagenicityTestData,Kirkland,D.J.,(cd)CambridgeUniversityPress,Cambridge,pp.141󰂗154.(14)Soper,K.A.andGalloway,S.M.(1994),ReplicateFlasksarenotNecessaryforInVitroChromosome

AberrationAssaysinCHOCells,MutationRes.,312,pp.139󰂗149.(15)Krahn,D.F.,Barsky,F.C.andMcCooey,K.T.(1982),CHO/HGPRTMutationAssay:Evaluationof

GasesandVolatileLiquids,in:Tice,R.R.,Costa,D.L.,Schaich,K.M.(eds),GenotoxicEffectsofAirborneAgents,NewYork,Plenum,pp.91󰂗103.(16)Zamora,P.O.,Benson,J.M.,Li,A.P.andBrooks,A.L.(1983),EvaluationofanExposureSystem

UsingCellsGrownonCollagenGelsforDetectingHighlyVolatileMutagensintheCHO/HGPRTMutationAssay,EnvironmentalMutagenesis,5,pp.795󰂗801.(17)Locke-Huhle,C.(1983),EndoreduplicationinChinesehamstercellsduringalpha-radiationinducedG2

arrest,MutationRes.,119,pp.403󰂗413.(18)Huang,Y.,Change,C.andTrosko,J.E.(1983),Aphidicolin-inducedendoreduplicationinChinese

hamstercells,CancerRes.,43,pp.1362󰂗13.󰂒

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ANNEX4B

󰂑B.11.MUTAGENICITY󰂗INVIVOMAMMALIANBONEMARROWCHROMOSOMEABERRATIONTEST

1.METHOD

ThismethodisareplicateoftheOECDTG475,MammalianBoneMarrowChromosomeAberrationTest(1997).

1.1.INTRODUCTION

Themammalianinvivochromosomeaberrationtestisusedforthedetectionofstructuralchromosomeaberrationsinducedbythetestsubstancetothebonemarrowcellsofanimals,usuallyrodents(1)(2)(3)(4).Structuralchromosomeaberrationsmaybeoftwotypes,chromosomeorchromatid.Anincreaseinpolyploidymayindicatethatachemicalhasthepotentialtoinducenumericalaberrations.Withthemajorityofchemicalmutagens,inducedaberrationsareofthechromatid-type,butchromosome-typeaberrationsalsooccur.Chromosomemutationsandrelatedeventsarethecauseofhumangeneticdiseasesandthereissubstantialevidencethatchromosomemutationsandrelatedeventscausingalterationsinoncogenesandtumoursuppressergenesareinvolvedincancerinhumansandexperimentalsystems.

Rodentsarcroutinelyusedinthistest.Bonemarrowisthetargettissueinthistest,sinceitisahighlyvascularisedtissue,anditcontainsapopulationofrapidlycyclingcellsthatcanbereadilyisolatedandprocessed.Otherspeciesandtargettissuesarenotthesubjectofthismethod.

Thischromosomeaberrationtestisespeciallyrelevanttoassessingmutagenichazardinthatitallowsconsiderationoffactorsofinvivometabolism,pharmacokineticsandDNA-repairprocessesalthoughthesemayvaryamongspeciesandamongtissues.Aninvivotestisalsousefulforfurtherinvestigationofamutageniceffectdetectedbyaninvitrotest.

Ifthereisevidencethatthetestsubstance,orareactivemetabolite,willnotreachthetargettissue,itisnotappropriatetousethistest.SeealsoGeneralintroductionPartB.

1.2.DEFINITIONS

Chromatid-typeaberration:structuralchromosomedamageexpressedasbreakageofsinglechromatidsorbreakageandreunionbetweenchromatids.

Chromosome-typeaberration:structuralchromosomedamageexpressedasbreakage,orbreakageandreunion,ofbothchromatidsatanidenticalsite.

Endoreduplication:aprocessinwhichafteranSperiodofDNAreplication,thenucleusdoesnotgointomitosisbutstartsanotherSperiod.Theresultischromosomeswith4,8,16,󰂅chromatids.

Gap:anachromaticlesionsmallerthanthewidthofonechromatid,andwithminimummisalignmentofthechromatid(s).

Numericalaberration:achangeinthenumberofchromosomesfromthenormalnumbercharacteristicofthecellsutilised.

Polyploidy:amultipleofthehaploidchromosomenumber(n)otherthanthediploidnumber(i.e.3n,4nandsoon).

Structuralaberration:achangeinchromosomestructuredetectablebymicroscopicexaminationofthemetaphasestageofcelldivision,observedasdeletionsandfragments,intrachangesorinterchanges.

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1.3.

ENOfficialJournaloftheEuropeanCommunities8.6.2000

PRINCIPLEOFTHETESTMETHOD

Animalsareexposedtothetestsubstancebyanappropriaterouteofexposureandaresacrificedatappropriatetimesaftertreatment.Priortosacrifice,animalsaretreatedwithametaphasearrestingagent(e.g.colchicineorColcemid®).Chromosomepreparationsarethenmadefromthebonemarrowcellsandstained,andmetaphasecellsareanalysedforchromosomeaberrations.

1.4.DESCRIPTIONOFTHETESTMETHOD

1.4.1.Preparations

1.4.1.1.Selectionofanimalsspecies

Rats,miceandChinesehamstersarecommonlyused,althoughanyappropriatemammalianspeciesmaybeused.Commonlyusedlaboratorystrainsofyounghealthyadultanimalsshouldbeemployed.Atthecommencementofthestudy,theweightvariationofanimalsshouldbeminimalandnotexceed±20%ofthemeanweightofeachsex.

1.4.1.2.Housingandfeedingconditions

GeneralconditionsreferredintheGeneralIntroductiontoPartBareappliedalthoughtheaimforhumidityshouldbe50󰂖60%.

1.4.1.3.Preparationoftheanimals

Healthyyoungadultanimalsarerandomlyassignedtothecontrolandtreatmentgroups.Cagesshouldbearrangedinsuchawaythatpossibleeffectsduetocageplacementareminimised.Theanimalsareidentifieduniquely.Theanimalsareacclimatedtothelaboratoryconditionsforatleastfivedays.

1.4.1.4.Preparationofdoses

Solidtestsubstancesshouldbedissolvedorsuspendedinappropriatesolventsorvehiclesanddiluted,ifappropriate,priortodosingoftheanimals.Liquidtestsubstancesmaybedoseddirectlyordilutedpriortodosing.Freshpreparationsofthetestsubstanceshouldbeemployedunlessstabilitydatademonstratetheacceptabilityofstorage.

1.4.2.Testconditions

1.4.2.1.Solvent/vehicle

Thesolvent/vehicleshouldnotproducetoxiceffectsatthedoselevelsused,andshouldnotbesuspectedofchemicalreactionwiththetestsubstance.Ifotherthanwell-knownsolvents/vehiclesareused,theirinclusionshouldbesupportedwithdataindicatingtheircompatibility.Itisrecommendedthatwhereverpossible,theuseofanaqueoussolvent/vehicleshouldbeconsideredfirst.

1.4.2.2.Controls

Concurrentpositiveandnegative(solvent/vehicle)controlsshouldbeincludedforeachsexineachtest.Exceptfortreatmentwiththetestsubstance,animalsinthecontrolgroupsshouldbehandledinanidenticalmannertotheanimalsinthetreatedgroups.

Positivecontrolsshouldproducestructuralaberrationsinvivoatexposurelevelsexpectedtogiveadetectableincreaseoverbackground.Positivecontroldosesshouldbechosensothattheeffectsareclearbutdonotimmediatelyrevealtheidentityofthecodedslidestothereader.Itisacceptablethatthepositivecontrolbe

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administeredbyaroutedifferentfromthetestsubstanceandsampledatonlyasingletime.Theuseofchemicalclassrelatedpositivecontrolchemicalsmaybeconsidered,whenavailable.Examplesofpositivecontrolsubstancesinclude:

SubstanceCASNoEinecsNo

ethylmethansulphonateethylnitrosoureamitomycinCcyclophosphamide

cyclophosphamidemonohydratetriethylenemelamine

62-50-0759-73-950-07-750-18-06055-19-251󰂖18󰂖3

200-536-7212-072-2200-008-6200-015-4

200-083-5

Negativecontrols,treatedwithsolventorvehiclealone,andotherwisetreatedinthesamewayasthetreatmentgroups,shouldbeincludedforeverysamplingtime,unlessacceptableinter-animalvariabilityandfrequenciesofcellswithchromosomeaberrationsareavailablefromhistoricalcontroldata.Ifsinglesamplingisappliedfornegativecontrols,themostappropriatetimeisthefirstsamplingtime.Inaddition,untreatedcontrolsshouldalsobeusedunlesstherearehistoricalorpublishedcontroldatademonstratingthatnodeleteriousourmutageniceffectsareinducedbythechosensolvent/vehicle.

1.5.PROCEDURE

1.5.1.Numberandsexofanimals

Eachtreatedandcontrolgroupincludeatleastfiveanalysableanimalspersex.Ifatthetimeofthestudytherearedataavailablefromstudiesinthesamespeciesandusingthesamerouteofexposurethatdemonstratethattherearenosubstantialdifferencesintoxicitybetweensexes,thentestinginasinglesexwillbesufficient.Wherehumanexposuretochemicalsmaybesex-specific,asforexamplewithsomepharmaceuticalagents,thetestshouldbeperformedwithanimalsoftheappropriatesex.

1.5.2.Treatmentschedule

Testsubstancesarepreferablyadministeredasasingletreatment.Testsubstancesmayalsobeadministeredasasplitdose,i.e.twotreatmentsonthesamedayseparatedbynomorethanafewhours,tofacilitateadministeringalargevolumeofmaterial.Otherdoseregimensshouldbescientificallyjustified.

Samplesshouldbetakenattwoseparatetimesfollowingtreatmentononeday.Forrodents,thefirstsamplingintervalis1,5normalcellcyclelength(thelatterbeingnormally12󰂖18hours)followingtreatment.Sincethetimerequiredforuptakeandmetabolismofthetestsubstanceaswellasitseffectoncellcyclekineticscanaffecttheoptimumtimeforchromosomeaberrationdetection,alatersamplecollection24hoursafterthefirstsampletimeisrecommended.Ifdoseregimensofmorethanonedayareused,onesamplingtimeat1,5normalcellcyclelengthsafterthefinaltreatmentshouldbeused.

Priortosacrifice,animalsareinjectedintraperitoneallywithanappropriatedoseofametaphasearrestingagent(e.g.Colcemid®orcolchicine).Animalsaresampledatanappropriateintervalthereafter.Formicethisintervalisapproximately3󰂖5hours;forChinesehamstersthisintervalasapproximately4󰂖5hours.Cellsareharvestedfromthebonemarrowandanalysedforchromosomeaberrations.

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1.5.3.

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Ifarangefindingstudyisperformedbecausetherearenosuitabledataavailable,itshouldbeperformedinthesamelaboratory,usingthesamespecies,strain,sex,andtreatmentregimentobeusedinthemainstudy(5).Ifthereistoxicity,threedoselevelsareusedforthefirstsamplingtime.Thesedoselevelsshouldcoverarangefromthemaximumtolittleornotoxicity.Atthelatersamplingtimeonlythehighestdoseneedstobeused.Thehighestdoseisdefinedasthedose-producingsignsoftoxicitysuchthathigherdoselevels,basedonthesamedosingregimen,wouldbeexpectedtoproducelethality.Substanceswithspecificbiologicalactivitiesatlownon-toxicdoses(suchashormonesandmitogens)maybeexceptionstothedose-settingcriteriaandshouldbeevaluatedonacase-by-casebasis.Thehighestdosemayalsobedefinedasadosethatproducessomeindicationoftoxicityinthebonemarrow(e.g.greaterthan50%reductioninmitoticindex).

1.5.4.Limittest

Ifatestatonedoselevelofatleast2000mg/kgbodyweightusingasingletreatment,orastwotreatmentsonthesameday,producesnoobservabletoxiceffects,andifgenotoxicitywouldnotbeexpectedbasedondatafromstructurallyrelatedsubstances,thenafullystudyusingthreedoselevelsmaynotbeconsiderednecessary.Forstudiesofalongerduration,thelimitdoseis2000mg/kg/bodyweight/dayfortreatmentupto14days,and1000mg/kig/bodyweight/dayfortreatmentlongerthan14days.Expectedhumanexposuremayindicatetheneedforahigherdoseleveltobeusedinthelimittest.

1.5.5.Administrationofdoses

Thetestsubstanceisusuallyadministeredbygavageusingastomachtubeorasuitableintubationcannula,orbyintraperitonealinjection.Otherroutesofexposuremaybeacceptablewheretheycanbejustified.Themaximumvolumeofliquidthatcanbeadministeredbygavageorinjectionatonetimedependsonthesizeofthetestanimal.Thevolumeshouldnotexceed2ml/100gbodyweight.Theuseofvolumeshigherthanthesemustbejustified.Exceptforirritatingorcorrosivesubstanceswhichwillnormallyrevealexacerbatedeffectswithhigherconcentrations,variabilityintestvolumeshouldbeminimisedbyadjustingtheconcentrationtoensureaconstantvolumeatalldoselevels.

1.5.6.Chromosomepreparation

Immediatelyaftersacrifice,bonemarrowisobtained,exposedtohypotonicsolutionandfixed.Thecellsarethenspreadonslidesandstained.

1.5.7.Analysis

Themitoticindexshouldbedeterminedasameasureofcytotoxicityinatleast1000cellsperanimalforalltreatedanimals(includingpositivecontrols)anduntreatednegativecontrolanimals.

Atleast100cellsshouldbeanalysedforeachanimal.Thisnumbercouldbereducedwhenhighnumbersofaberrationsareobserved.Allslides,includingthoseofpositiveandnegativecontrols,shouldbeindependentlycodedbeforemicroscopicanalysis.Sinceslidepreparationproceduresoftenresultinthebreakageofaproportionofmetaphaseswithlossofchromosomes,thecellsscoredshouldthereforecontainanumberofcentromeresequaltothenumber2n±2.

2.DATA

2.1.TREATMENTOFRESULTS

Individualanimaldatashouldbepresentedintabularform.Theexperimentalunitistheanimal.Foreachanimalthenumberofcellsscored,thenumberofaberrationspercellandthepercentageofcellswithstructuralchromosomeaberration(s)shouldbeevaluated.Differenttypesofstructuralchromosomeaberrationsshouldbelistedwiththeirnumbersandfrequenciesfortreatedandcontrolgroups.Gapsarerecordedseparatelyandreportedbutgenerallynotincludedinthetotalaberrationfrequency.Ifthereisnoevidenceforadifferenceinresponsebetweenthesexes,thedatafrombothsexesmaybecombinedforstatisticalanalysis.

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EVALUATIONANDINTERPRETATIONOFRESULTS

Thereareseveralcriteriafordeterminingapositiveresult,suchasadose-relatedincreaseintherelativenumberofcellswithchromosomeaberrationsoraclearincreaseinthenumberofcellswithaberrationsinasingledosegroupatasinglesamplingtime.Biologicalrelevanceoftheresultsshouldbeconsideredfirst.Statisticalmethodsmaybeusedasanaidinevaluatingthetestresults(6).Statisticalsignificanceshouldnotbetheonlydeterminingfactorforapositiveresponse.Equivocalresultsshouldbeclarifiedbyfurthertestingpreferablyusingamodificationofexperimentalconditions.

Anincreaseinpolyploidymayindicatethatthetestsubstancehasthepotentialtoinducenumericalchromosomeaberrations.Anincreaseinendoreduplicationmayindicatethatthetestsubstancehasthepotentialtoinhibitcellcycleprogression(7)(8).

Atestsubstanceforwhichtheresultsdonotmeettheabovecriteriaisconsiderednon-mutagenicinthistest.Althoughmostexperimentswillgiveclearlypositiveornegativeresults,inrarecasesthedatasetwillprecludemakingadefinitejudgementabouttheactivityofthetestsubstance.Resultsmayremainequivocalorquestionableregardlessofthenumberofexperimentsperformed.

Positiveresultsfromtheinvivochromosomeaberrationtestindicatethatasubstanceinduceschromosomeaberrationsinthebonemarrowofthespeciestested.Negativeresultsindicatethat,underthetestconditions,thetestsubstancedoesnotinducechromosomeaberrationsinthebonemarrowofthespeciestested.Thelikelihoodthatthetestsubstanceoritsmetabolitesreachthegeneralcirculationorspecificallythetargettissue(e.g.systemictoxicity)shouldbediscussed.

3.REPORTINGTESTREPORT

Testtestreportmustincludethefollowinginformation:Solventvehicle:

󰂗justificationforchoiceofvehicle,

󰂗solubilityandstabilityofthetestsubstanceinsolvent/vehicle,ifknown.Testanimals:

󰂗species/strainused,

󰂗number,ageandsexofanimals,󰂗source,housingconditions,diet,etc.,

󰂗individualweightoftheanimalsatthestartofthetest,includingbodyweightrange,meansandstandard

deviationforeachgroup.Testconditions:

󰂗positiveandnegative(vehicle/solvent)controls,󰂗datafromrange-findingstudy,ifconducted,󰂗rationaleforcloselevelselection,󰂗detailsoftestsubstancepreparation,

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󰂗detailsoftheadministrationofthetestsubstance,󰂗rationaleforrouteofadministration,

󰂗methodsforverifyingthatthetestsubstancereachedthegeneralcirculationortargettissue,ifapplicable,󰂗conversionfromdiet/drinkingwatertestsubstanceconcentration(ppm)totheactualdose(mg/kgbody

weight/day),ifapplicable,󰂗detailsoffoodandwaterquality,

󰂗detaileddescriptionoftreatmentandsamplingschedules,󰂗methodsformeasurementsoftoxicity,

󰂗identityofmetaphasearrestingsubstance,itsconcentrationanddurationoftreatment,󰂗methodsofslidepreparation,󰂗criteriaforscoringaberrations,󰂗numberofcellsanalysedperanimal,

󰂗criteriaforconsideringstudiesaspositive,negativeorequivocal.Results:

󰂗signsoftoxicity,󰂗mitoticindex,

󰂗typeandnumberofaberrations,givenseparatelyforeachanimal,󰂗totalnumberofaberrationspergroupwithmeansandstandarddeviations,󰂗numberofcellswithaberrationspergroupwithmeansandstandarddeviations,󰂗changesinploidy,ifseen,

󰂗dose-responserelationship,wherepossible,󰂗statisticalanalyses,ifany,󰂗concurrentnegativecontroldata,

󰂗historicalnegativecontroldatawithranges,meansandstandarddeviations,󰂗concurrentpositivecontroldata.Discussionoftheresults.Conclusions.

4.REFERENCES(1)

Adler,I.D.(1984),CytogeneticTestsinMammals,in:MutagenicityTesting:aPracticalApproach,S.VenittandJ.M.Parry(eds),IRLPress,Oxford,WashingtonD.C.,pp.275󰂖306.

Preston,R.J.,Dean,B.J.,Galloway,S.,Holden,H.,McFee,A.F.andShelby,M.(1987),MammalianInVivoCytogeneticAssays:AnalysisofChromosomeAberrationsinBoneMarrowCells,MutationRes.,1,pp.157󰂖165.

(2)

8.6.2000EN(3)

OfficialJournaloftheEuropeanCommunities

Richold,M.,Chandley,A.,Ashby,J.,Gatehouse,D.G.,Bootman,J.andHenderson,L.(1990),InvivoCytogeneticAssays,in:D.J.Kirkland(ed.),BasicMutagenicityTests,UKEMSRecommendedProcedures.UKEMSSub-CommitteeonGuidelinesforMutagenicityTesting.Report,PartIrevised,CambridgeUniversityPress,Cambridge,NewYork,PortChester,Melbourne,Sydney,pp.115󰂖141.

Tice,R.R.,Hayashi,M.,MacGregaro,J.T.,Anderson,D.,Blakey,D.H.,Holden,H.E.,Kirsch-Volders,M.,OlesonJr.,F.B.,Pacchierotti,F.,Preston,R.J.,Romagna,F.,Shimada,H.,Sutou,S.andVannier,B.(1994),ReportfromtheWorkingGroupontheinVivoMammalianBoneMarrowChromosomalAberrationTest,MutationRes.,312,pp.305󰂖312.

Fielder,R.J.,Allen,J.A.,Boobis,A.R.,Botham,P.A.,Doe,J.,Esdaile,D.J.,Gatehouse,D.G.,Hodson-Walker,G.,Morton,D.B.,Kirkland,D.J.andRichold,M.(1992),ReportofBritishToxicologySociety/UKEnvironmentalMutagenSocietyWorkingGroup:DosesettinginInVivoMutagenicityAssays,Mutagenesis,7,pp.313󰂖319.

Lovell,D.P.,Anderson,D.,Albanese,R.,Amphlett,G.E.,Clare,G.,Ferguson,R.,Richold,M.,Papworth,D.G.andSavage,J.R.K.(19),StatisticalAnalysisofInVivoCytogeneticAssays,in:UKEMSSub-CommitteeonGuidelinesforMutagenicityTesting,ReportPartIII.StatisticalEvaluationofMutagenicityTestData,D.J.Kirkland(ed.)CambridgeUniversityPress,Cambridge,pp.184󰂖232.Locke-Huhle,C.(1983),EndoreduplicationinChinesehamstercellsduringalpha-radiation-inducedG2arrest,MutationRes.,119,pp.403󰂖413.

Huang,Y.,Change,C.andTrosko,J.E.(1983),Aphidicolin-inducedendoreduplicationinChinesehamstercells,CancerRes.,43,pp.1362󰂖13.󰂒

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(4)

(5)

(6)

(7)(8)

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ANNEX4C

󰂑B.12.MUTAGENICITY󰂗INVIVOMAMMALIANERYTHROCYTEMICRONUCLEUSTEST

1.METHOD

ThismethodisareplicateoftheOECDTG474,MammalianErythrocyteMicronucleusTest(1997).

1.1.INTRODUCTION

Themammalianinvivomicronucleustestisusedforthedetectionofdamageinducedbythetestsubstancetothechromosomesorthemitoticapparatusoferythroblastsbyanalysisoferythrocytesassampledinbonemarrowand/orperipheralbloodcellsofanimals,usuallyrodents.

Thepurposeofthemicronucleustestistoidentifysubstancesthatcausecytogeneticdamagewhichresultsintheformationofmicronucleicontaininglaggingchromosomefragmentsorwholechromosomes.

Whenabonemarrowerythroblastdevelopsintoapolychromaticerythrocyte,themainnucleusisextruded;anymicronucleusthathasbeenformedmayremainbehindintheotherwiseanucleatedcytoplasm.Visualisationofmicronucleiisfacilitatedinthesecellsbecausetheylackamainnucleus.Anincreaseinthefrequencyofmicronucleatedpolychromaticerythrocytesintreatedanimalsisanindicationofinducedchromosomedamage.

Thebonemarrowofrodentsisroutinelyusedinthistestsincepolychromaticerythrocytesareproducedinthattissue.Themeasurementofmicronucleatedimmature(polychromatic)erythrocytesinperipheralbloodisequallyacceptableinanyspeciesinwhichtheinabilityofthespleentoremovemicronucleatederythrocyteshasbeendemonstrated,orwhichhasshownanadequatesensitivitytodetectagentsthatcausestructuralornumericalchromosomeaberrations.Micronucleicanbedistinguishedbyanumberofcriteria.TheseincludeidentificationofthepresenceorabsenceofakinetochoreorcentromericDNAinthemicronuclei.Thefrequencyofmicronucleatedimmature(polychromatic)erythrocytesistheprincipalendpoint.Thenumberofmature(normochromatic)erythrocytesintheperipheralbloodthatcontainmicronucleiamongagivennumberofmatureerythrocytescanalsobeusedastheendpointoftheassaywhenanimalsaretreatedforfourweeksormore.

Thismammalianinvivomicronucleustestespeciallyrelevanttoassessingmutagenichazardinthatitallowsconsiderationoffactorsofinvivometabolism,pharmacokineticsandDNArepairprocessesalthoughthesemayvaryamongspecies,amongtissuesandamonggeneticendpoints.Aninvivoassayisalsousefulforfurtherinvestigationofamutageniceffectdetectedbyaninvitrosystem.

Ifthereisevidencethatthetestsubstance,orareactivemetabolite,willnotreachthetargettissue,itisnotappropriatetousethistest.SeealsoGeneralIntroductionPartB.

1.2.DEFINITIONS

Centromere(Kinetochore):region(s)ofachromosomewithwhichspindlefibersareassociatedduringcelldivision,allowingorderlymovementofdaughterchromosomestothepolesofthedaughtercells.

Micronuclei:smallnuclei,separatefromandadditionaltothemainnucleiofcells,producedduringtelophaseofmitosis(meiosis)bylaggingchromosomefragmentsorwholechromosomes.

Normochromaticerythrocyte:matureerythrocytethatlacksribosomesandcanbedistinguishedfromimmature,polychromaticerythrocytesbystainsselectiveforribosomes.

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Polychromaticerythrocyte:immatureerythrocyte,inanintermediatestageofdevelopment,thatstillcontainsribosomesandthereforecanbedistinguishedfrommature,normochromaticerythrocytesbystainsselectiveforribosomes.

1.3.PRINCIPLEOFTHETESTMETHOD

Animalsareexposedtothetestsubstancebyanappropriateroute.Ifbonemarrowisused,theanimalsaresacrificedatappropriatetimesaftertreatment,thebonemarrowextracted,andpreparationsmadeandstained(1)(2)(3)(4)(5)(6)(7).Whenperipheralbloodisused,thebloodiscollectedatappropriatetimesaftertreatmentandsmearpreparationsaremadeandstained(4)(8)(9)(10).Forstudieswithperipheralblood,aslittletimeaspossibleshouldelapsebetweenthelastexposureandcellharvest.Preparationsareanalysedforthepresenceofmicronuclei.

1.4.1.4.1.1.4.1.1.

DESCRIPTIONOFTHETESTMETHODPreparations

Selectionofanimalspecies

Miceorratsarerecommendedifbonemarrowisused,althoughanyappropriatemammalianspeciesmaybeused.Whenperipheralbloodisused,micearerecommended.However,anyappropriatemammalianspeciesmaybeusedprovideditisaspeciesinwhichthespleendoesnotremovemicronucleatederythrocytesoraspecieswhichhasshownanadequatesensitivitytodetectagentsthatcausestructuralornumericalchromosomeaberrations.Commonlyusedlaboratorystrainsofyounghealthyanimalsshouldbeemployed.Atthecommencementofthestudy,theweightvariationofanimalsshouldbeminimalandnotexceed±20%ofthemeanweightofeachsex.

1.4.1.2.Housingandfeedingconditions

GeneralconditionsreferredintheGeneralIntroductiontoPartBareappliedalthoughtheaimforhumidityshouldbe50󰂖60%.

1.4.1.3.Preparationoftheanimals

Healthyyoungadultanimalsarerandomlyassignedtothecontrolandtreatmentgroups.Theanimalsareidentifieduniquely.Theanimalsareacclimatedtothelaboratoryconditionsforatleastfivedays.Cagesshouldbearrangedinsuchawaythatpossibleeffectsduetocageplacementareminimised.

1.4.1.4.Preparationofdoses

Solidtestsubstancesshouldbedissolvedorsuspendedinappropriatesolventsorvehiclesanddiluted,ifappropriate,priortodosingoftheanimals.Liquidtestsubstancesmaybedoseddirectlyordilutedpriortodosing.Freshpreparationsofthetestsubstanceshouldbeemployedunlessstabilitydatademonstratetheacceptabilityofstorage.

1.4.2.1.4.2.1.

TestconditionsSolvent/vehicle

Thesolvent/vehicleshouldnotproducetoxiceffectsatthedoselevelsused,andshouldnotbesuspectedofchemicalreactionwiththetestsubstance.Ifotherthanwell-knownsolvents/vehiclesareused,theirinclusionshouldbesupportedwithreferencedataindicatingtheircompatibility.Itisrecommendedthatwhereverpossible,theuseofaqueoussolvent/vehicleshouldbeconsideredfirst.

1.4.2.2.Controls

Concurrentpositiveandnegative(solvent/vehicle)controlsshouldbeincludedforeachsexineachtest.Exceptfortreatmentwiththetestsubstance,animalsinthecontrolgroupsshouldbehandledinanidenticalmannertoanimalsofthetreatmentgroups.

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Positivecontrolsshouldproducemicronucleiinvivoatexposurelevelsexpectedtogiveadetectableincreaseoverbackground.Positivecontroldosesshouldbechosensothattheeffectsareclearbutdonotimmediatelyrevealtheidentityofthecodedslidestothereader.Itisacceptablethatthepositivecontrolbeadministeredbyaroutedifferentfromthetestsubstanceandsampledatonlyasingletime.Inaddition,theuseofchemicalclass-relatedpositivecontrolchemicalsmaybeconsidered,whenavailable.Examplesofpositivecontrolsubstancesinclude:

Substance

CASNo

EinecsNo

ethylmethansulphonateN-ethyl-N-nitrosoureamitomycinCcyclophosphamide

cyclophosphamidemonohydratetriethylenemelamine

62-50-0759-73-950-07-750-18-06055-19-251󰂖18󰂖3

200-536-7212-072-2200-008-6200-015-4

200-083-5

Negativecontrols,treatedwithsolventorvehiclealone,andotherwisetreatedinthesamewayasthetreatmentgroupsshouldbeincludedforeverysamplingtime,unlessacceptableinter-animalvariabilityandfrequenciesofcellswithmicronucleiaredemonstratedbyhistoricalcontroldata.Ifsinglesamplingisappliedfornegativecontrols,themostappropriatetimeisthefirstsamplingtime.Inaddition,untreatedcontrolsshouldalsobeusedunlesstherearehistoricalorpublishedcontroldatademonstratingthatnodeleteriousormutageniceffectsareinducedbythechosensolvent/vehicle.

Ifperipheralbloodisused,apre-treatmentsamplemayalsobeacceptableasaconcurrentnegativecontrol,butonlyintheshortperipheralbloodstudies(e.g.1󰂖3treatment(s))whentheresultingdataareintheexpectedrangeforthehistoricalcontrol.

1.5.PROCEDURE

1.5.1.Numberandsexofanimals

Eachtreatedandcontrolgroupmustincludeatleastfiveanalysableanimalspersex(11).Ifatthetimeofthestudytherearedataavailablefromstudiesinthesamespeciesandusingthesamerouteofexposurethatdemonstratethattherearenosubstantialdifferencesbetweensexesintoxicity,thentestinginasinglesexwillbesufficient.Wherehumanexposuretochemicalsmaybesex-specific,asforexamplewithsomepharmaceuticalagents,thetestshouldbeperformedwithanimalsoftheappropriatesex.

1.5.2.Treatmentschedule

Nostandardtreatmentschedule(i.e.one,twoormoretreatmentsat24-hourintervals)canberecommended.Thesamplesfromextendeddoseregimensareacceptableaslongasapositiveeffecthasbeendemonstratedforthisstudyor,foranegativestudy,aslongastoxicityhasbeendemonstratedorthelimitdosehasbeenused,anddosingcontinueduntilthetimeofsampling.Testsubstancesmayalsobeadministeredasasplitdose,i.e.twotreatmentsonthesamedayseparatedbynomorethanafewhours,tofacilitateadministeringalargevolumeofmaterial.

Thetestmaybeperformedintwoways:

(a)animalsaretreatedwiththetestsubstanceonce.Samplesofbonemarrowaretakenatleasttwice,

startingnotearlierthan24hoursaftertreatment,butnotextendingbeyond48hoursaftertreatmentwithappropriateintervalsbetweensamples.Theuseofsamplingtimesearlierthan24hoursaftertreatmentshouldbejustified.Samplesofperipheralbloodaretakenatleasttwice,startingnotearlier

8.6.2000ENOfficialJournaloftheEuropeanCommunities

than36hoursaftertreatment,withappropriateintervalsfollowingthefirstsample,butnotextendingbeyond72hours.Whenapositiveresponseisrecognisedatonesamplingtime,additionalsamplingisnotrequired;

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(b)Iftwoormoredailytreatmentsareused(e.g.twoormoretreatmentsat24-hourintervals),samples

shouldbecollectedoncebetween18and24hoursfollowingthefinaltreatmentforthebonemarrowandoncebetween36and48hoursfollowingthefinaltreatmentfortheperipheralblood(12).Othersamplingtimesmaybeusedinaddition,whenrelevant.

1.5.3.Doselevels

Ifarangefindingstudyisperformedbecausetherearenotsuitabledataavailable,itshouldbeperformedinthesamelaboratory,usingthesamespecies,strain,sex,andtreatmentregimentobeusedinthemainstudy(13).Ifthereistoxicity,threedoselevelsareusedforthefirstsamplingtime.Thesedoselevelsshouldcoverarangefromthemaximumtolittleornotoxicity.Atthelatersamplingtimeonlythehighestdoseneedstobeused.Thehighestdoseisdefinedasthedoseproducingsignsoftoxicitysuchthathigherdoselevels,basedonthesamedosingregimen,wouldbeexpectedtoproducelethality.Substanceswithspecificbiologicalactivitiesatlownon-toxicdoses(suchashormonesandmitogens)maybeexceptionstothedose-settingcriteriaandshouldbeevaluatedonacase-by-casebasis.Thehighestdosemayalsobedefinedasadosethatproducessomeindicationoftoxicityinthebonemarrow(e.g.areductionintheproportionofimmatureerythrocytesamongtotalerythrocytesinthebonemarroworperipheralblood).

1.5.4.Limittest

Ifatestatonedoselevelofatleast2000mg/kgbodyweightusingasingletreatment,orastwotreatmentsonthesameday,producesnoobservabletoxiceffects,andifgenotoxicitywouldnotbeexpectedbasedupondatafromstructurallyrelatedsubstances,thenafullystudyusingthreedoselevelsmaynotbeconsiderednecessary.Forstudiesofalongerduration,thelimitdoseis2000mg/kg/bodyweight/dayfortreatmentupto14days,and1000mg/kg/bodyweight/dayfortreatmentlongerthan14days.Expectedhumanexposuremayindicatetheneedforahigherdoseleveltobeusedinthelimittest.

1.5.5.Administrationofdoses

Thetestsubstanceisusuallyadministeredbygavageusingastomachtubeorasuitableintubationcannula,orbyintraperitonealinjection.Otherroutesofexposuremaybeacceptablewheretheycanbejustified.Themaximumvolumeofliquidthatcanbeadministeredbygavageorinjectionatonetimedependsonthesizeofthetestanimal.Thevolumeshouldnotexceed2ml/100gbodyweight.Theuseofvolumeshighercanthesemustbejustified.Exceptforirritatingorcorrosivesubstanceswhichwillnormallyrevealexacerbatedeffectswithhigherconcentrations,variabilityintestvolumeshouldbeminimisedbyadjustingtheconcentrationtoensureaconstantvolumeatalldoselevels.

1.5.6.Bonemarrow/bloodpreparation

Bonemarrowcellsareusuallyobtainedfromthefemursortibiasimmediatelyfollowingsacrifice.Commonly,cellsareremovedfromfemursortibias,preparedandstainedusingestablishedmethods.Peripheralbloodisobtainedfromthetailveinorotherappropriatebloodvessel.Bloodcellsareimmediatelystainedsupravitally(8)(9)(10)orsmearpreparationsaremadeandthenstained.TheusedofaDNAspecificstain(e.g.acridineorange(14)orHoechst33258pluspyronin-Y(15))caneliminatesomeoftheartifactsassociatedwithusinganon-DNA-specificstain.Thisadvantagedoesnotprecludetheuseofconventionalstains(e.g.,Giemsa).Additionalsystems(e.g.cellulosecolumnstoremovenucleatedcells(16))canalsobeusedprovidedthatthesesystemshavebeenshowntoadequatelyworkformicronucleuspreparationinthelaboratory.

1.5.7.Analysis

Theproportionofimmatureamongtotal(immature+mature)erythrocytesisdeterminedforeachanimalbycountingatotalatleast200erythrocytesforbonemarrowand1000erythrocytesforperipheralblood(17).Allslides,includingthoseofpositiveandnegativecontrols,shouldbeindependentlycodedbeforemicroscopicanalysis.Atleast2000immatureerythrocytesperanimalarescoredfortheincidenceof

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micronucleatedimmatureerythrocytes.Additionalinformationmaybeobtainedbyscoringmatureerythrocytesformicronuclei.Whenanalysingslides,theproportionofimmatureerythrocytesamongtotalerythrocytesshouldnotbelessthan20%ofthecontrolvalue.Whenanimalsaretreatedcontinuouslyforfourweeksormore,atleast2000matureerythrocytesperanimalcanalsobescoredfortheincidenceofmicronuclei.Systemsforautomatedanalysis(imageanalysisandflowcytometricanalysisofcellsuspensions)areacceptablealternativestomanualevaluationifappropriatelyjustifiedandvalidated.

2.DATA

2.1.TREATMENTOFRESULTS

Individualanimaldatashouldbepresentedintabularform.Theexperimentalunitistheanimal.Thenumberofimmatureerythrocytesscored,thenumberofmicronucleatedimmatureerythrocytes,andthenumberofimmatureamongtotalerythrocytesshouldbelistedseparatelyforeachanimalanalysed.Whenanimalsaretreatedcontinuouslyforfourweeksormore,thedataonmatureerythrocytesshouldalsobegivenifitiscollected.Theproportionofimmatureamongtotalerythrocytesand,ifconsideredapplicable,thepercentageofmicronucleatederythrocytesisgivenforeachanimal.Ifthereisnoevidenceforadifferenceinresponsebetweenthesexes,thedatafrombothsexesmaybecombinedforstatisticalanalysis.

2.2.EVALUATIONANDINTERPRETATIONOFRESULTS

Thereareseveralcriteriafordeterminingapositiveresult,suchasadose-relatedincreaseinthenumberofmicronucleatedcellsoraclearincreaseinthenumberofmicronucleatedcellsinasingledosegroupatasinglesamplingtime.Biologicalrelevanceoftheresultsshouldbeconsideredfirst.Statisticalmethodsmaybeusedasanaidinevaluatingthetestresults(18)(19).Statisticalsignificanceshouldnotbeonlydeterminingfactorforapositiveresponse.Equivocalresultsshouldbeclarifiedbyfurthertestingpreferablyusingamodificationofexperimentalconditions.

Atestsubstanceforwhichtheresultsdonotmeettheabovecriteriaisconsiderednon-mutagenicinthistest.Althoughmostexperimentswillgiveclearlypositiveornegativeresults,inrarecasesthedatasetwillprecludemakingadefinitejudgementabouttheactivityofthetestsubstance.Resultsmayremainequivocalorquestionableregardlessofthenumberoftimestheexperimentisrepeated.

Positiveresultsinthemicronucleustestindicatethatthesubstanceinducesmicronucleiwhicharetheresultofchromosomaldamageordamagetothemitoticapparatusintheerythroblastsofthetestspecies.Negativeresultsindicatethat,underthetestconditions,thetestsubstancedoesnotproducemicronucleiintheimmatureerythrocytesofthetestspecies.

Thelikelihoodthatthetestsubstanceoritsmetabolitesreachthegeneralcirculationorspecificallythetargettissue(e.g.systemictoxicity)shouldbediscussed.

3.REPORTINGTESTREPORT

Thetestreportshouldincludethefollowinginformation:Solvent/vehicle:

󰂗justificationforchoiceofvehicle,

󰂗solubilityandstabilityofthetestsubstanceinsolvent/vehicle,ifknown.

8.6.2000ENTestanimals:

󰂗species/strainused,

OfficialJournaloftheEuropeanCommunitiesL136/55

󰂗number,ageandsexofanimals,󰂗source,housingconditions,diet,etc.,

󰂗individualweightoftheanimalsatthestartofthetest,includingbodyweightrange,meanandstandard

deviationforeachgroup.Testconditions:

󰂗positiveandnegative(vehicle/solvent)controldata,󰂗datafromrange-findingstudy,ifconducted,󰂗rationalefordoselevelselection,󰂗detailsoftestsubstancepreparation,

󰂗detailsoftheadministrationofthetestsubstance,󰂗rationaleforrouteofadministration,

󰂗methodsforverifyingthatthetestsubstancereachedthegeneralcirculationortargettissue,ifapplicable,󰂗conversionfromdiet/drinkingwatertestsubstanceconcentration(ppm)totheactualdose(mg/kgbody

weight/day),ifapplicable,󰂗detailsoffoodandwaterquality,

󰂗detaileddescriptionoftreatmentandsamplingschedules,󰂗methodsofslidepreparation,󰂗methodsformeasurementsoftoxicity,

󰂗criteriaforscoringmicronucleatedimmatureerythrocytes,󰂗numberofcellsanalysedperanimal,

󰂗criteriaforconsideringstudiesaspositive,negativeorequivocal.Results:

󰂗signsoftoxicity,

󰂗proportionofimmatureerythrocytesamongtotalerythrocytes,

󰂗numberofmicronucleatedimmatureerythrocytes,givenseparatelyforeachanimal,󰂗mean±standarddeviationofmicronucleatedimmatureerythrocytespergroup,󰂗dose-responserelationship,wherepossible,󰂗statisticalanalysesandmethodsapplied,󰂗concurrentandhistoricalnegativedata,󰂗concurrentpositivecontroldata.Discussionoftheresults.Conclusions.

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4.

ENREFERENCES

OfficialJournaloftheEuropeanCommunities8.6.2000

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(3)Heddle,J.A.,Salamone,M.F.,Hite,M.,Kirkhart,B.,Mavournin,K.,MacGregor,J.G.andNewell,G.W.

(1983),TheInductionofMicronucleiasaMeasureofGenotoxicity,MutationRes.123,pp.61󰂖118.(4)Mavournin,K.H.,Blakey,D.H.,Cimino,M.C.,Salamone,M.F.andHeddle,J.A.(1990),TheInVivo

MicronucleusAssayinMammalianBoneMarrowandPeripheralBlood.AreportoftheU.S.EnvironmentalProtectionAgencyGene-ToxProgram,MutationRes.,239,pp.29󰂖80.(5)MacGregor,J.T.,Schlegel,R.,Choy,W.N.andWehr,C.M.(1983),MicronucleiinCirculating

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Wild,D.(1987),GuidelinesfortheConductofMicronucleusAssaysinMammalianBoneMarrowErythrocytes,MutationRes.,1,pp.103󰂖112.(7)MacGregor,J.T.,Wehr,C.M.,Henika,P.R.andShelby,M.E.(1990),TheinvivoErythrocyte

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Hodson-Walker,G.,Morton,D.B.,Kirkland,D.J.andRochold,M.(1992),ReportofBritishToxicologySociety/UKEnvironmentalMutagenSocietyWorkingGroup:DoseSettinginInVivoMutagenicityAssays,Mutagenesis,7,pp.313󰂖319.(14)Hayashi,M.,Sofumi,T.andIshidate,M.Jr.(1983),AnApplicationofAcridineOrangeFluorescent

StainingtotheMicronucleusTest,MutationRes.,120,pp.241󰂖247.(15)MacGregor,J.T.,Wehr,C.M.andLanglois,R.G.(1983),ASimpleFluorescentStainingProcedurefor

MicronucleiandRNAinErythrocytesUsingHoechst33258andPyrominY,MutationRes.,120,pp.269󰂖275.(16)Romagna,F.andStaniforth,C.D.(19),Theautomatedbonemarrowmicronucleustest,Mutation

Res.,213,pp.91󰂖104.(17)Gollapudi,B.andMcFadden,L.G.(1995),Samplesizefortheestimationofpolychromaticto

normochromaticerythrocyteratiointhebonemarrowmicronucleustest,MutationRes.,347,pp.97󰂖99.(18)Richold,M.,Ashby,J.,Bootman,J.,Chandley,A.,Gatehouse,D.G.andHenderson,L.(1990),InVivo

CytogeneticsAssay,in:D.J.Kirkland(ed.),BasicMutagenicitytests,UKEMSRecommendedProcedures,UKEMSSub-CommitteeonGuidelinesforMutagenicityTesting.Report,Part1,revised,CambridgeUniversityPress,Cambridge,NewYork,PortChester,Melbourne,Sydney,pp.115󰂖141.(19)Lovell,D.P.,Anderson,D.,Albanese,R.,Amphlett,G.E.,Clare,G.,FergusonR.,Richold,M.,

Papworth,D.G.andSavage,J.R.K.(19),StaticicalAnalysisofInVivoCytogeneticAssays,in:D.J.Kirkland(ed.),StatisticalEvaluationofMutagenicityTestData.UKEMSSub-CommitteeonGuidelinesforMutagenicityTesting.Report,PartIII,CambridgeUniversityPress,Cambridge,NewYork,PortChester,Melbourne,Sydney,pp.184󰂖232.󰂒

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ANNEX4D

󰂑B.13/14.MUTAGENICITY󰂗REVERSEMUTATIONTESTBACTERIA

1.METHOD

ThismethodisareplicateoftheOECDTG471,BacterialReverseMutationTest(1997).

1.1.INTRODUCTION

Thebacterialreversemutationtestusesamino-acidrequiringstrainsofSalmonellatyphimuriumandEscherichiacolitodetectpointmutations,whichinvolvesubstitution,additionordeletionofoneorafewDNAbasepairs(1)(2)(3).Theprincipleofthisbacterialreversemutationtestisthatitdetectsmutationswhichrevertmutationspresentintheteststrainsandrestorethefunctionalcapabilityofthebacteriatosynthesiseanessentialaminoacid.Therevertantbacteriaaredetectedbytheirabilitytogrowintheabsenceoftheamino-acidrequiredbytheparentteststrain.

Pointmutationsarethecauseofmanyhumangeneticdiseasesandthereissubstantialevidencethatpointmutationsinoncogenesandtumoursuppressorgenesofsomaticcellsareinvolvedintumourformationinhumansandexperimentalanimals.Thebacterialreversemutationtestisrapid,inexpensiveandrelativelyeasytoperform.ManyoftheteststrainshaveseveralfeaturesthatmakethemmoresensitiveforthedetectionofmutationsincludingresponsiveDNAsequencesatthereversionsites,increasedcellpermeabilitytolargemoleculesandeliminationofDNArepairsystemsorenhancementoferror-proneDNArepairprocesses.Thespecificityoftheteststrainscanprovidesomeusefulinformationonthetypesofmutationsthatareinducedbygenotoxicagents.Averylargedatabaseofresultsforawidevarietyofstructuresisavailableforbacterialreversemutationtestsandwell-establishedmethodologieshavebeendevelopedfortestingchemicalswithdifferentphysico-chemicalproperties,includingvolatilecompounds.SeealsoGeneralIntroductionPartB.

1.2.DEFINITIONS

AreversemutationtestineitherSalmonellatyphimuriumorEscherichiacolidetectsmutationinanaminoacidrequiringstrain(histidineortryptophan,respectively)toproduceastrainindependentofanoutsidesupplyofaminoacid.

BasepairsubstitutionmutagensareagentsthatcauseabasechangeinDNA.Inareversiontestthischangemayoccuratthesiteoftheoriginalmutation,oratasecondsiteinthebacterialgenome.

FrameshiftmutagensareagentsthatcausetheadditionordeletionofoneormorebasepairsintheDNA,thuschangingthereadingframeintheRNA.

1.3.INITIALCONSIDERATIONS

Thebacterialreversemutationtestutilisesprokaryoticcells,whichdifferfrommammaliancellsinsuchfactorsasuptake,metabolism,chromosomestructureandDNArepairprocesses.Testsconductedinvitrogenerallyrequiretheuseofanexogenoussourceofmetabolicactivation.Invitrometabolicactivationsystemscannotmimicentirelythemammalianinvivoconditions.Thetestthereforedoesnotprovidedirectinformationonthemutagenicandcarcinogenicpotencyofasubstanceinmammals.

Thebacterialreversemutationtestiscommonlyemployedasaninitialscreenforgenoloxicactivityand,inparticular,forpointmutation-inducingactivity.Anextensivedatabasehasdemonstratedthatmanychemicalsthatarepositiveinthistestalsoexhibitmutagenicactivityinothertests.Thereareexamplesofmutagenicagentswhicharenotdetectedbythistest;reasonsfortheseshortcomingscanbeascribedtothe

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specificnatureoftheendpointdetected,differencesinmetabolicactivation,ordifferencesinbioavailability.Ontheotherhand,factorswhichenhancethesensitivityofthebacterialreversemutationtestcanleadtoanoverestimationofmutagenicactivity.

Thebacterialreversemutationtestmaynotbeappropriatefortheevaluationofcertainclassesofchemicals,forexamplehighlybactericidalcompounds(e.g.certainantibiotics)andthosewhicharethought(orknown)tointerferespecificallywiththemammaliancellreplicationsystem(e.g.sometopoisomeraseinhibitorsandsomenucleosideanalogues).Insuchcases,mammalianmutationtestsmaybemoreappropriate.

Althoughmanycompoundsthatarepositiveinthistestaremammaliancarcinogens,thecorrelationisnotabsolute.Itisdependentonchemicalclassandtherearecarcinogensthatarenotdetectedbythistestbecausetheyactthroughother,non-genotoxic,mechanismsormechanismsabsentinbacterialcells.

1.4.PRINCIPLEOFTHETESTMETHOD

Suspensionsofbacterialcellsareexposedtothetestsubstanceinthepresenceandintheabsenceofanexogenousmetabolicactivationsystem.Intheplateincorporationmethod,thesesuspensionsaremixedwithanoverlayagarandplatedimmediatelyontominimalmedium.Inthepreincubationmethod,thetreatmentmixtureisincubatedandthenmixedwithanoverlayagarbeforeplatingontominimalmedium.Forbothtechniques,aftertwoorthreedaysofincubation,revertantcoloniesarecountedandcomparedtothenumberofspontaneousrevertantcoloniesonsolventcontrolplates.

Severalproceduresforperformingthebacterialreversemutationtesthavebeendescribed.Amongthosecommonlyusedaretheplateincorporationmethod(1)(2)(3)(4),thepreincubationmethod(2)(3)(5)(6)(7)(8),thefluctuationmethod(9)(10),andthesuspensionmethod(11).Modificationsforthetestingofgasesorvapourshavebeendescribed(12).

Theproceduresdescribedinthemethodpertainprimarilytotheplateincorporationandpreincubationmethods.Eitherofthemisacceptableforconductingexperimentsbothwithandwithoutmetabolicactivation.Somesubstancesmaybedetectedmoreefficientlyusingthepreincubationmethod.Thesesubstancesbelongtochemicalclassesthatincludeshortchainaliphaticnitrosamines,divalentmetals,aldehydes,azo-dyesanddiazocompounds,pyrollizidinealkaloids,allylcompoundsandnitrocompounds(3).Itisalsorecognisedthatcertainclassesofmutagensarenotalwaysdetectedusingstandardproceduressuchastheplateincorporationmethodorpreincubationmethod.Theseshouldberegardedas󰂓specialcases󰂔anditisstronglyrecommendedthatalternativeproceduresshouldbeusedfortheirdetection.Thefollowing󰂓specialcases󰂔couldbeidentified(togetherwithexamplesofproceduresthatcouldbeusedfortheirdetection):azo-dyesanddiazocompounds(3)(5)(6)(13),gasesandvolatilechemicals(12)(14)(15)(16)andglycosides(17)(18).Adeviationfromthestandardprocedureneedstobescientificallyjustified.

1.5.DESCRIPTIONOFTHETESTMETHOD

1.5.1.Preparations

1.5.1.1.Bacteria

Freshculturesofbacteriashouldbegrownuptothelateexponentialorearlystationaryphaseofgrowth(approximately109cellsperml).Culturesinlatestationaryphaseshouldnotbeused.Itisessentialthattheculturesusedintheexperimentcontainahightitreofviablebacteria.Thetitremaybedemonstratedeitherfromhistoricalcontroldataongrowthcurves,orineachassaythroughthedeterminationofviablecellnumbersbyaplatingexperiment.

Therecommendedincubationtemperatureis37°C.

Atleastfivestrainsofbacteriashouldbeused.TheseshouldincludefourstrainsofS.typhimurium(TA1535;TA1537orTA97aorTA97;TA98;andTA100)thathavebeenshowntobereliableandreproduciblyresponsivebetweenlaboratories.ThesefourS.typhimuriumstrainshaveGCbasepairsattheprimaryreversionsiteanditisknownthattheymaynotdetectcertainoxidisingmutagens,cross-linkingagentsandhydrazines.SuchsubstancesmaybedetectedbyE.coliWP2strainsorS.typhimuriumTA102(19)whichhaveanATbasepairattheprimaryreversionsite.Thereforetherecommendedcombinationofstrainsis:

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󰂗S.typhimuriumTA1535,and

󰂗S.typhimuriumTA1537orTA97orTA97a,and󰂗S.typhimuriumTA98,and󰂗S.typhimuriumTA100,and

󰂗E.coliWP2uvrA,orE.coliWP2uvrA(pKM101),orS.typhimuriumTA102.

Inordertodetectcross-linkingmutagensitmaybepreferabletoincludeTA102ortoaddaDNArepair-proficientstrainofE.coli(e.g.E.coliWP2orE.coliWP2(pKM101)).

Establishedproceduresforstockculturepreparation,markerverificationandstorageshouldbeused.Theamino-acidrequirementforgrowthshouldbedemonstratedforeachfrozenstockculturepreparation(histidineforS.typhimuriumstrains,andtryptophanforE.colistrains).Otherphenotypiccharacteristicsshouldbesimilarlychecked,namely:thepresenceorabsenceofR-factorplasmidswhereappropriate(i.e.ampicillinresistanceinstrainsTA98,TA100,andTA97aorTA97,WP2uvrAandWP2uvrA(pKM101),andampicillin+tetracyclineresistanceinstrainTA102);thepresenceofcharacteristicmutations(i.e.rfamutationinS.typhimuriumthroughsensitivitytocrystalviolet,anduvrAmutationinE.colioruvrBmutationinS.typhimurium,throughsensitivitytoultra-violetlight)(2)(3).Thestrainsshouldalsoyieldspontaneousrevertantcolonyplatecountswithinthefrequencyrangesexpectedfromthelaboratory'shistoricalcontroldataandpreferablywithintherangereportedintheliterature.

1.5.1.2.

Medium

Anappropriateminimalagar(e.g.containingVogel-BonnerminimalmediumEandglucose),andanoverlayagarcontaininghistidineandbiotinortryptophantoallowforafewcelldivisions,isused(1)(2)(9).

1.5.1.3.

Metabolicactivation

Bacteriashouldbeexposedtothetestsubstancebothinthepresenceandabsenceofanappropriatemetabolicactivationsystem.Themostcommonlyusedsystemisacofactor-supplementedpost-mitochondrialfraction(S9)preparedfromtheliversofrodentstreatedwithenzyme-inducingagentssuchasAroclor1254(1)(2)oracombinationofphenobarbitoneandb-naphthoflavone(18)(20)(21).Thepost-mitochondrialfractionisusuallyusedatconcentrationsintherangefrom5to30%v/vintheS9-mix.Thechoiceandconditionofametabolicactivationsystemmaydependupontheclassofchemicalbeingtested.Insomecases,itmaybeappropriatetoutilisemorethanoneconcentrationofpost-mitochondrialfraction.Forazo-dyesanddiazo-compounds,usingareductivemetabolicactivationsystemmaybemoreappropriate(6)(13).

1.5.1.4.

Testsubstance/preparation

Solidtestsubstancesshouldbedissolvedorsuspendedinappropriatesolventsorvehiclesanddilutedifappropriatepriortotreatmentofthebacteria.Liquidtestsubstancesmaybeaddeddirectlytothetestsystemsand/ordilutedpriortotreatment.Freshpreparationsshouldbeemployedunlessstabilitydatademonstratetheacceptabilityofstorage.

Thesolvent/vehicleshouldnotbesuspectedofchemicalreactionwiththetestsubstanceandshouldbecompatiblewiththesurvivalofthebacteriaandtheS9activity(22).Ifotherthanwell-knownsolvent/vehiclesareused,theirinclusionshouldbesupportedbydataindicatingtheircompatibility.Itisrecommendedthatwhereverpossible,theuseofanaqueoussolvent/vehiclebeconsideredfirst.Whentestingwater-unstablesubstances,theorganicsolventsusedshouldbefreeofwater.

1.5.2.1.5.2.1.1.5.2.2.

TestconditionsTeststrains(see1.5.1.1)Exposureconcentration

Amongstthecriteriatobetakenintoconsiderationwhendeterminingthehighestamountofthetestsubstancetobeusedarethecytotoxicityandthesolubilityinthefinaltreatmentmixture.

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Itmaybeusefultodeterminetoxicityandinsolubilityinapreliminaryexperiment.Cytotoxicitymaybedetectedbyareductioninthenumberofrevertantcolonies,aclearingordiminutionofthebackgroundlawn,orthedegreeofsurvivaloftreatedcultures.Thecytotoxicityofasubstancemaybealteredinthepresenceofmetabolicactivationsystems.Insolubilityshouldbeassessedasprecipitationinthefinalmixtureundertheactualtestconditionsandevidenttotheunaidedeye.

Therecommendedmaximumtestconcentrationforsolublenon-cytotoxicsubstancesis5mg/plateor5µl/plate.Fornon-cytotoxicsubstancesthatarenotsolubleat5mg/plateor5µl/plate,oneormoreconcentrationstestedshouldbeinsolubleinthefinaltreatmentmixture.Testsubstancesthatarecytotoxicalreadybelow5mg/plateor5µl/plateshouldbetesteduptoacytotoxicconcentration.Theprecipitateshouldnotinterferewiththescoring.

Atleastfivedifferentanalysableconcentrationsofthetestsubstanceshouldbeusedwithapproximatelyhalflog(i.e.Ó10)intervalsbetweentestpointsforaninitialexperiment.Smallerintervalsmaybeappropriatewhenaconcentration-responseisbeinginvestigated.Testingabovetheconcentrationof5mg/plateor5µl/platemaybeconsideredwhenevaluatingsubstancescontainingsubstantialamountsofpotentiallymutagenicimpurities.

1.5.2.3.Negativeandpositivecontrols

Concurrentstrain-specificpositiveandnegative(solventorvehicle)controls,bothwithandwithoutmetabolicactivation,shouldbeincludedineachassay.Positivecontrolconcentrationsthatdemonstratetheeffectiveperformanceofeachassayshouldbeselected.

Forassaysemployingametabolicactivationsystem,thepositivecontrolreferencesubstance(s)shouldbeselectedonthebasisofthetypeofbacteriastrainsused.

Thefollowingsubstancesareexamplesofsuitablepositivecontrolsforassayswithmetabolicactivation:

Substance

CASNo

EinecsNo

9,10-dimethylanthracene7,12-dimethylbenz[a]anthracenebenzo[a]pyrene2-aminoanthracenecyclophosphamide

cyclophosphamidemonohydrate

781-43-157-97-650-32-8613-13-850-18-06055-19-2

212-308-4200-359-5200-028-5210-330-9200-015-4

Thefollowingsubstanceisasuitablepositivecontrolforthereductivemetabolicactivationmethod:

Substance

CASNo

EinecsNo

CongoRed573-58-0209-358-4

2-AminoanthraceneshouldnotbeusedasthesoleindicatoroftheefficacyoftheS9-mix.If2-aminoanthraceneisused,eachbatchofS9shouldalsobecharacterisedwithamutagenthatrequiresmetabolicactivationbymicrosomalenzymes,e.g.,benzo[a]pyrene,dimethylbenzanthracene.

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Thefollowingsubstancesareexamplesofstrain-specificpositivecontrolsforassaysperformedwithoutexogenousmetabolicactivationsystem:

Substance

CASNo

EinecsNo

Strain

sodiumazide2-nitrofluorene9-aminoacridineICR191

cumenehydroperoxidemitomycinC

N-ethyl-N-nitro-N-nitrosoguanidine

26628-22-8607-57-0-45-917070-45-080-15-950-07-770-25-7

247-852-1210-138-5201-995-6241-129-4201-254-7200-008-6200-730-1

TA1535andTA100

TA98

TA1537,TA97andTA97aTA1537,TA97andTA97a

TA102

WP2uvrAandTA102WP2,WP2uvrAandWP2uvrA(pKM101)WP2,WP2uvrAandWP2uvrA(pKM101)plasmid-containingstrains

4-nitroquinoline-1-oxide56-57-5200-281-1

furylfuramide(AF2)

3688-53-7

Otherappropriatepositivecontrolreferencesubstancesmaybeused.Theuseofchemicalclass-relatedpositivecontrolchemicalsshouldbeconsidered,whenavailable.

Negativecontrols,consistingofsolventorvehiclealone,withouttestsubstance,andotherwisetreatedinthesamewayasthetreatmentgroups,shouldbeincluded.Inaddition,untreatedcontrolsshouldalsobeusedunlesstherearehistoricalcontroldatademonstratingthatnodeleteriousormutageniceffectsareinducedbythechosensolvent.

1.5.3.Procedure

Fortheplateincorporationmethod(1)(2)(3)(4),withoutmetabolicactivation,usually0,05mlor0,1mlofthetestsolutions,0,1mloffreshbacterialculture(containingapproximately108viablecells)and0,5mlofsterilebufferaremixedwith2,0mlofoverlayagar.Fortheassaywithmetabolicactivation,usually0,5mlofmetabolicactivationmixturecontaininganadequateamountofpost-mitochondrialfraction(intherangefrom5to30%v/vinthemetabolicactivationmixture)aremixedwiththeoverlayagar(2,0ml),togetherwiththebacteriaandtestsubstance/testsolution.Thecontentsofeachtubearemixedandpouredoverthesurfaceofaminimalagarplate.Theoverlayagarisallowedtosolidifybeforeincubation.

Forthepreincubationmethod(2)(3)(5)(6),thetestsubstance/testsolutionispreincubatedwiththeteststrain(containingapproximately108viablecells)andsterilebufferorthemetabolicactivationsystem(0,5ml)usuallyfor20minutesormoreat30󰂖37°Cpriortomixingwiththeoverlayagarandpouringontothesurfaceofaminimalagarplate.Usually0,05or0,1mloftestsubstance/testsolution,0,1mlofbacteriaand0,5mlofS9-mixorsterilebufferaremixedwith2,0mlofoverlayagar.Tubesshouldbeaeratedduringpre-incubationbyusingashaker.

Foranadequateestimateofvariation,triplicateplatingshouldbeusedateachdoselevel.Theuseofduplicateplatingisacceptablewhenscientificallyjustified.Theoccasionallossofaplatedoesnotnecessarilyinvalidatetheassay.

Gaseousorvolatilesubstancesshouldbetestedbyappropriatemethods,suchasinsealedvessels(12)(14)(15)(16).

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1.5.4.

ENIncubation

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Allplatesinagivenassayshouldbeincubatedat37°Cfor48󰂖72hours.Aftertheincubationperiod,thenumberofrevertantcoloniesperplateiscounted.

2.DATA

2.1.TREATMENTOFRESULTS

Datashouldbepresentedasthenumberofrevertantcoloniesperplate.Thenumberofrevertantcoloniesonbothnegative(solventcontrol,anduntreatedcontrolifused)andpositivecontrolplatesshouldalsobegiven.Individualplatecounts,themeannumberofrevertantcoloniesperplateandthestandarddeviationshouldbepresentedforthetestsubstanceandpositiveandnegative(untreatedand/orsolvent)controls.

Thereisnorequirementforverificationofaclearpositiveresponse.Equivocalresultsshouldbeclarifiedbyfurthertestingpreferablyusingamodificationofexperimentalconditions.Negativeresultsneedtobeconfirmedonacase-by-casebasis.Inthosecaseswhereconfirmationofnegativeresultsisnotconsiderednecessary,justificationshouldbeprovided.Modificationofstudyparameterstoextendtherangeofconditionsassessedshouldbeconsideredinfollow-upexperiments.Studyparametersthatmightbemodifiedincludetheconcentrationspacing,themethodoftreatment(plate-incorporationorliquidpre-incubation),andmetabolicactivationconditions.

2.2.EVALUATIONANDINTERPRETATIONOFRESULTS

Thereareseveralcriteriafordeterminingapositiveresult,suchasaconcentration-relatedincreaseovertherangetestedand/orareproducibleincreaseatoneormoreconcentrationsinthenumberofrevertantcoloniesperplateinatleastonestrainwithorwithoutmetabolicactivationsystem(23).Biologicalrelevanceoftheresultsshouldbeconsideredfirst.Statisticalmethodsmaybeusedasanaidinevaluatingthetestresults(24).However,statisticalsignificanceshouldnotbetheonlydeterminingfactorforapositiveresponse.

Atestsubstanceforwhichtheresultsdonotmeettheabovecriteriaisconsiderednon-mutagenicinthistest.Althoughmostexperimentswillgiveclearlypositiveornegativeresults,inrarecasesthedatasetwillprecludemakingadefinitejudgementabouttheactivityofthetestsubstance.Resultsmayremainequivocalorquestionableregardlessofthenumberoftimestheexperimentisrepeated.

PositiveresultsfromthebacterialreversemutationtestindicatethatthesubstanceinducespointmutationsbybasesubstitutionsorframeshiftsinthegenomeofeitherSalmonellatyphimuriumand/orEscherichiacoli.Negativeresultsindicatethatunderthetestconditions,thetestsubstanceisnotmutagenicinthetestedspecies.

3.REPORTINGTESTREPORT

Thetestreportmustincludethefollowinginformation:Solvent/vehicle:

󰂗justificationforchoiceofsolvent/vehicle,

󰂗solubilityandstabilityofthetestsubstanceinsolvent/vehicle,ifknown.Strains:󰂗strainsused,

󰂗numberofcellsperculture,󰂗straincharacteristics.

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󰂗amountoftestsubstanceperplate(mg/plateorµl/plate)withrationaleforselectionofdoseandnumber

ofplatesperconcentration,󰂗mediaused,

󰂗typeandcompositionofmetabolicactivationsystem,includingacceptabilitycriteria,󰂗treatmentprocedures.Results:

󰂗signsoftoxicity,󰂗signsofprecipitation,󰂗individualplatecounts,

󰂗themeannumberofrevertantcoloniesperplateandstandarddeviation,󰂗dose-responserelationship,wherepossible,󰂗statisticalanalyses,ifany,

󰂗concurrentnegative(solvent/vehicle)andpositivecontroldata,withranges,meansandstandard

deviations,󰂗historicalnegative(solvent/vehicle)andpositivecontroldatawithranges,meansandstandarddeviations.Discussionofresults.Conclusions.

4.REFERENCES

(1)Ames,B.N.,McCann,J.andYamasakiE.(1975),MethodsofDetectingCarcinogensandMutagenswith

theSalmonella/Mammalian-MicrosomeMutagenicityTest,MutationRes.,31,pp.347󰂖3.(2)Maron,D.M.andAmes,B.N.(1983),RevisedMethodsfortheSalmonellaMutagenicityTest,Mutation

Res.,113,pp.173󰂖215.(3)Gatehouse,D.,Haworth,S.,Cebula,T.,Gocke,E.,Kier,L.,Matsushima,T.,Melcion,C.,Nohmi,T.,

Venitt,S.andZeiger,E.(1994),RecommendationsforthePerformanceofBacterialMutationAssays,MutationRes.,312,pp.217󰂖233.(4)Kier,L.D.,BrusickD.J.,Auletta,A.E.,VonHalle,E.S.,Brown,M.M.,Simmon,V.F.,Dunkel,V.,

McCann,J.,Mortelmans,K.,Prival,M.,Rao,T.K.andRayV.(1986),TheSalmonellatyphimurium/MammalianMicrosomalAssay:AReportoftheU.S.EnvironmentalProtectionAgencyGene-ToxProgram,MutationRes.,168,pp.69󰂖240.(5)Yahagi,T.,Degawa,M.,Seino,Y.Y.,Matsushima,T.,Nagao,M.,Sugimura,T.andHashimoto,Y.

(1975),MutagenicityofCarcinogenAzoDyesandtheirDerivatives,CancerLetters1,pp.91󰂖96.(6)Matsushima,M.,Sugimura,T.,Nagao,M.,Yahagi,T.,Shirai,A.andSawamura,M.(1980),Factors

ModulatingMutagenicityMicrobialTests,in:Short-termTestSystemsforDetectingCarcinogens,ed.NorpothK.H.andGarner,R.C.,Springer,Berlin-Heidelberg-NewYork,pp.273󰂖285.(7)Gatehouse,D.G.,Rowland,I.R.,Wilcox,P.,Callender,R.D.andFoster,R.(1980),BacterialMutation

Assays,in:BasicMutagenicityTests:UKEMSPart1Revised,ed.D.J.Kirkland,CambridgeUniversityPress,pp.13󰂖61.(8)Aeschacher,H.U.,Wolleb,U.andPorchet,L.(1987),LiquidPreincubationMutagenicityTestfor

Foods,J.FoodSafety,8,pp.167󰂖177.

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(9)Green,M.H.L.,Muriel,W.J.andBridges,B.A.(1976),Useofasimplifiedfluctuationtesttodetect

lowlevelsofmutagens,MutationRes.,38,pp.33󰂖42.(10)Hubbard,S.A.,Green,M.H.L.,Gatehouse,D.andBridges,J.W.(1984),TheFluctuationTestin

Bacteria,in:HandbookofMutagenicityTestProcedures,2ndEdition,ed.Kilbey,B.J.,Legator,M.,Nichols,W.andRamel,C.,Elsevier,Amsterdam-NewYork-Oxford,pp.141󰂖161.(11)Thompson,E.D.andMelampy,P.J.(1981),AnExaminationoftheQuantitativeSuspensionAssayfor

MutagenesiswithStrainsofSalmonellatyphimurium,EnvironmentalMutagenesis,3,pp.453󰂖465.(12)Araki,A.,Noguchi,T.,Kato,F.andMatsushima,T.(1994),ImprovedMethodforMutagenicityTesting

ofGaseousCompoundsbyUsingaGasSamplingBag,MutationRes.,307,pp.335󰂖344.(13)Prival,M.J.,Bell,S.J.,Mitchell,V.D.,Reipert,M.D.andVaughan,V.L.(1984),Mutagenicityof

BenzidineandBenzidine-CongenerDyesandSelectedMonoazoDyesinaModifiedSalmonellaAssay,MutationRes.,136,pp.33󰂖47.(14)Zeiger,E.,Anderson,B.E.,Haworth,S.,Lawlor,T.andMortelmans,K.(1992),SalmonellaMutagenicity

Tests.V.ResultsfromtheTestingof311Chemicals,Environ.Mol.Mutagen.,19,pp.2󰂖141.(15)Simmon,V.,Kauhanen,K.andTardiff,R.G.(1977),MutagenicActivityofChemicalsIdentifiedin

DrinkingWater,inProgressinGeneticToxicology,D.Scott,B.BridgesandF.,Sobels(eds.)Elsevier,Amsterdam,pp.249󰂖258.(16)Hughes,T.J.,Simmons,D.M.,Monteith,I.G.andClaxton,L.D.(1987),VaporisationTechniqueto

MeasureMutagenicActivityofVolatileOrganicChemicalsintheAmes/SalmonellaAssay,EnvironmentalMutagenesis,9,pp.421󰂖441.(17)Matsushima,T.,Matsumoto,A.,Shirai,M.,Sawamura,M.,andSugimura,T.(1979),Mutagenicityof

theNaturallyOccurringCarcinogenCycasinandSyntheticMethylazoxyMethaneConjugatesinSalmonellatyphimurium,CancerRes.,39,pp.3780󰂖3782.(18)Tamura,G.,Gold,C.,Ferro-Luzzi,A.andAmes,B.N.(1980),Fecalase:AModelforActivationof

DietaryGlycosidestoMutagensbyIntestinalFlora,Proc.Natl.Acad.Sci.USA,77,pp.4961󰂖4965.(19)Wilcox,P.,Naidoo,A.,Wedd,D.J.andGatehouse,D.G.(1990),ComparisonofSalmonellatyphimurium

TA102withEscherichiacoliWP2Testerstrains,Mutagenesis,5,pp.285󰂖291.(20)Matsushima,T.,Sawamura,M.,Hara,K.andSugimura,T.(1976),ASafeSubstituteforPolychlorinated

BiphenylsasanInducerorMetabolicActivationSystems,in:InvitroMetabolicActivationinMutagenesisTesting,eds.F.J.deSerresetal.Elsevier,NorthHolland,pp.85󰂖88.(21)Elliot,B.M.,Combes,R.D.,Elcombe,C.R.,Gatehouse,D.G.,Gibson,G.G.,Mackay,J.M.andWolf,

R.C.(1992),AlternativestoAroclor1254-inducedS9ininvitroGenotoxicityAssays,Mutagenesis,7,pp.175󰂖177.(22)Maron,D.,Katzenellenbogen,J.andAmes,B.N.(1981),CompatibilityofOrganicSolventswiththe

Salmonella/MicrosomeTest,MutationRes.,88,pp.343󰂖350.(23)Claxton,L.D.,Allen,J.,Auletta,A.,Mortelmans,K.,Nestmann,E.andZeiger,E.(1987),Guideforthe

Salmonellatyphimurium/MammalianMicrosomeTestsforBacterialMutagenicity,MutationRes.,1,pp.83󰂖91.(24)Mahon,G.A.T.,Green,M.H.L.,Middleton,B.,Mitchel,I.,Robinson,W.D.andTweats,D.J.(19),

AnalysisofDatafromMicrobialColonyAssays,in:UKEMSSub-CommitteeonGuidelinesforMutagenicityTesting,PartII.StatisticalEvaluationofMutagenicityTestData,ed.Kirkland,D.J.,CambridgeUniversityPress,pp.28󰂖65.󰂒

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ANNEX4E

󰂑B.17.MUTAGENICITY󰂗INVITROMAMMALIANCELLGENEMUTATIONTEST

1.METHOD

ThismethodisareplicateoftheOECDTG476,InVitroMammalianCellGeneMutationTest(1997).

1.1.INTRODUCTION

Theinvitromammaliancellgenemutationtestcanbeusedtodetectgenemutationsinducedbychemicalsubstances.SuitablecelllinesincludeL5178Ymouselymphomacells,theCHO,CHO-AS52andV79linesofChinesehamstercells,andTK6humanlymphoblastoidcells(1).Inthesecelllinesthemostcommonlyusedgeneticendpointsmeasuremutationatthymidinekinase(TK)andhypoxanthine-guaninephosphoribosyltransferase(HPRT),andatransgeneofxanthine-guaninephosphoribosyltransferase(XPRT).TheTK,HPRTandXPRTmutationtestsdetectdifferentspectraofgeneticevents.TheautosomallocationofTKandXPRTmayallowthedetectionofgeneticevents(e.g.largedeletions)notdetectedattheHPRTlocusonXchromosomes(2)(3)(4)(5)(6).

Intheinvitromammaliancellgenemutationtest,culturesofestablishedcelllinesorcellstrainscanbeused.Thecellsusedareselectedonthebasisofgrowthabilityincultureandstabilityofthespontaneousmutationfrequency.

Testsconductedinvitrogenerallyrequiretheuseofanexogenoussourceofmetabolicactivation.Thismetabolicactivationsystemcannotmimicentirelythemammalianinvivoconditions.Careshouldbetakentoavoidconditionswhichwouldleadtoresultsnotreflectingintrinsicmutagenicity.PositiveresultswhichdonotreflectintrinsicmutagenicitymayarisefromchangesinpH,osmolalityorhighlevelsofcytotoxicity(7).Thistestisusedtoscreenforpossiblemammalianmutagensandcarcinogens.Manycompoundsthatarepositiveinthistestaremammaliancarcinogens;however,thereisnotaperfectcorrelationbetweenthistestandcarcinogenicity.Correlationisdependentonchemicalclassandthereisincreasingevidencethattherearecarcinogensthatarenotdetectedbythistestbecausetheyappeartoactthroughother,non-genotoxicmechanismsormechanismsabsentinbacterialcells(6).SeealsoGeneralIntroductionPartB:

1.2.DEFINITIONS

Forwardmutation:agenemutationfromtheparentaltypeofthemutantformwhichgivesrisetoanalterationoralossoftheenzymaticactivityofthefunctionoftheencodedprotein.

Basepairsubstitutionmutagens:substanceswhichcausesubstitutionofoneorseveralbasepairsintheDNA.

Frameshiftmutagens:SubstanceswhichcausetheadditionordeletionofsingleormultiplebasepairsintheDNAmolecule.

Phenotypicexpressiontime:aperiodduringwhichunalteredgeneproductsaredepletedfromnewlymutatedcells.

Mutantfrequency:thenumberofmutantcellsobserveddividedbythenumberofviablecells.

Relativetotalgrowth:increaseincellnumberovertimecomparedtoacontrolpopulationofcells;calculatedastheproductofsuspensiongrowthrelativetothenegativecontroltimescloningefficiencyrelativetonegativecontrol.

Relativesuspensiongrowth:increaseincellnumberovertheexpressionperiodrelativetothenegativecontrol.

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Viability:thecloningefficiencyofthetreatedcellsatthetimeofplatinginselectiveconditionsaftertheexpressionperiod.

Survival:thecloningefficiencyofthetreatedcellswhenplatedattheendofthetreatmentperiod;survivalisusuallyexpressedinrelationtothesurvivalofthecontrolcellpopulation.

1.3.PRINICIPLEOFTHETESTMETHOD

Cellsdeficientinthymidinekinase(TK)duetothemutationTK+/¯RTK¯/¯areresistanttothecytotoxiceffectsofthepyrimidineanaloguetrifluorothymidine(TFT).ThymidinekinaseproficientcellsaresensitivetoTFT,whichcausestheinhibitionofcellularmetabolismandhaltsfurthercelldivision.ThusmutantcellsareabletoproliferateinthepresenceofTFT,whereasnormalcells,whichcontainthymidinekinase,arenot.Similarly,cellsdeficientinHPRTorXPRTareselectedbyresistanceto6-thioguanine(TG)or8-azaguanine(AG).Thepropertiesofthetestsubstanceshouldbeconsideredcarefullyifabaseanalogueoracompoundrelatedtotheselectiveagentistestedinanyofthemammaliancellgenemutationtests.Forexample,anysuspectedselectivetoxicitybythetestsubstanceformutantandnon-mutantcellsshouldbeinvestigated.Thus,performanceoftheselectionsystem/agentmustbeconfirmedwhentestingchemicalsstructurallyrelatedtotheselectiveagent(8).

Cellsinsuspensionormonolayercultureareexposedtothetestsubstance,bothwithandwithoutmetabolicactivation,forasuitableperiodoftimeandsubculturedtodeterminecytotoxicityandtoallowphenotypicexpressionpriortomutantselection(9)(10)(11)(12)(13).Cytotoxicityisusuallydeterminedbymeasuringtherelativecloningefficiency(survival)orrelativetotalgrowthoftheculturesafterthetreatmentperiod.Thetreatedculturesaremaintainedingrowthmediumforasufficientperiodoftime,characteristicofeachselectedlocusandcelltype,toallownear-optimalphenotypicexpressionofinducedmutations.Mutantfrequencyisdeterminedbyseedingknownnumbersofcellsinmediumcontainingtheselectiveagenttodetectmutantcellsandinmediumwithoutselectiveagenttodeterminethecloningefficiency(viability).Afterasuitableincubationtime,coloniesarecounted.Themutantfrequencyisderivedfromthenumberofmutantcoloniesinselectivemediumandthenumberofcoloniesinnon-selectivemedium.

1.4.DESCRIPTIONOFTHETESTMETHOD

1.4.1.Preparations

1.4.1.1.Cells

AvarietyofcelltypesareavailableforuseinthistestincludingsubclonesofL5171Y,CHO,CHO-AS52,V79orTK6cells.Celltypesusedinthistestshouldhaveademonstratedsensitivitytochemicalmutagens,ahighcloningefficiencyandastablespontaneousmutantfrequency.Cellsshouldbecheckedformycoplasmacontaminationandshouldnotbeusedifcontaminated.

Thetestshouldbedesignedtohaveapredeterminedsensitivityandpower.Thenumberofcells,culturesandconcentrationsoftestsubstanceusedshouldreflectthesedefinedparameters(14).Theminimalnumberofviablecellssurvivingtreatmentandusedateachstageinthetestshouldbebasedonthespontaneousmutationfrequency.Ageneralguideistouseacellnumberwhichisatleast10timestheinverseofthespontaneousmutationfrequency.However,itisrecommendedtoutiliseatleast106cells.Adequatehistoricaldataonthecellsystemusedshouldbeavailabletoindicateconsistentperformanceofthetest.

1.4.1.2.Mediaandcultureconditions

Appropriateculturemedia,andincubationconditions(culturevessels,temperature,CO2concentrationandhumidity)shouldbeused.Mediashouldbechosenaccordingtotheselectivesystemsandcelltypeusedinthetest.Itisparticularlyimportantthatcultureconditionsshouldbechosenthatensureoptimalgrowthofcellsduringtheexpressionperiodandcolonyformingabilityofbothmutantandnon-mutantcells.

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1.4.1.3.

ENPreparationofcultures

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Cellsarepropagatedfromstockcultures,seededinculturemediumandincubatedat37°C.Priortouseinthistest,culturesmayneedtobecleansedofpre-existingmutantcells.

1.4.1.4.

Metabolicactivation

Cellsshouldbeexposedtothetestsubstancebothinthepresenceandabsenceofanappropriatemetabolicactivationsystem.Themostcommonlyusedsystemisacofactor-supplementedpost-mitochondrialfraction(S9)preparedfromtheliversofrodentstreatedwithenzyme-inducingagentssuchasAroclor1254(15)(16)(17)(18)oracombinationofphenobarbitoneandb-naphthoflavone(19)(20).

Thepost-mitochondrialfractionisusuallyusedatconcentrationsintherangefrom1󰂗10%v/vinthefinaltestmedium.Thechoiceandconditionofametabolicactivationsystemmaydependupontheclassofchemicalbeingtested.Insomecasesitmaybeappropriatetoutilisemorethanoneconcentrationofpost-mitochondrialfraction.

Anumberofdevelopments,includingtheconstructionofgeneticallyengineeredcelllinesexpressingspecificactivatingenzymes,mayprovidethepotentialforendogenousactivation.Thechoiceofthecelllinesusedshouldbescientificallyjustified(e.g.bytherelevanceofthecytochromeP450isoenzymeforthemetabolismofthetestsubstance).

1.4.1.5.

Testsubstancepreparation

Solidtestsubstancesshouldbedissolvedorsuspendedinappropriatesolventsorvehiclesanddilutedifappropriatepriortotreatmentofthecells.Liquidtestsubstancesmaybeaddeddirectlytothetestsystemsand/ordilutedpriortotreatment.Freshpreparationsofthetestsubstanceshouldbeemployedunlessstabilitydatademonstratetheacceptabilityofstorage.

1.4.2.1.4.2.1.

TestconditionsSolvent/vehicle

Thesolvent/vehicleshouldnotbesuspectedofchemicalreactionwiththetestsubstanceandshouldbecompatiblewiththesurvivalofthecellsandtheS9activity.Ifotherthanwell-knownsolvent/vehiclesareused,theirinclusionshouldbesupportedbydataindicatingtheircompatibility.Itisrecommendedthatwhereverpossible,theuseofanaqueoussolvent/vehiclebeconsideredfirst.Whentestingwater-unstablesubstances,theorganicsolventsusedshouldbefreeofwater.Watercanberemovedbyaddingamolecularsieve.

1.4.2.2.

Exposureconcentrations

Amongthecriteriatobeconsideredwhendeterminingthehighestconcentrationarecytotoxicity,solubilityinthetestsystemandchangesinpHorosmolality.

Cytotoxicityshouldbedeterminedwithandwithoutmetabolicactivationinthemainexperimentusinganappropriateindicationofcellintegrityandgrowth,suchasrelativecloningefficiency(survival)orrelativetotalgrowth.Itmaybeusefultodeterminecytotoxicityandsolubilityinapreliminaryexperiment.

Atlastfouranalysableconcentrationsshouldbeused.Wherethereiscytotoxicity,theseconcentrationsshouldcoverarangefromthemaximumtolittleornotoxicity;thiswillusuallymeanthattheconcentrationlevelsshouldbeseparatedbynomorethanafactorbetween2andÓ10.Ifthemaximumconcentrationisbasedoncytotoxicitythenitshouldresultinapproximately10󰂗20%(butnotlessthan10%)relativesurvival(relativecloningefficiency)orrelativetotalgrowth.Forrelativelynon-cytotoxicsubstances,themaximumtestconcentrationshouldbe5mg/ml,5µl/ml,or1,01M,whicheveristhelowest.

Relativelyinsolublesubstancesshouldbetesteduptoorbeyondtheirlimitofsolubilityundercultureconditions.Evidenceofinsolubilityshouldbedeterminedinthefinaltreatmentmediumtowhichcellsareexposed.Itmaybeusefultoassesssolubilityatthebeginningandtheendofthetreatment,assolubilitycanchangeduringthecourseofexposureinthetestsystemduetopresenceofcells,S9,serumetc.Insolubilitycanbedetectedbyusingtheunaidedeye.Theprecipitateshouldnotinterferewiththescoring.

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1.4.2.3.

ENControls

OfficialJournaloftheEuropeanCommunities8.6.2000

Concurrentpositiveandnegative(solventorvehicle)controls,bothwithandwithoutmetabolicactivationshouldbeincludedineachexperiment.Whenmetabolicactivationisused,thepositivecontrolchemicalshouldbetheonethatrequiresactivationtogiveamutagenicresponse.Examplesofpositivecontrolsubstancesinclude:

Metabolicactivationcondition

Locus

Substance

CASNo

EinecsNo

Absenceofexogenousmetabolicactivation

HPRTEthylmethansulphonateEthylnitrosourea

62-50-0759-73-966-27-3

200-536-7212-072-2200-625-0

TK(smallandlargecolonies)

XPRT

Methylmethansulphonate

EthylmethanesulphonateEthylnitrosourea

62-50-0759-73-956-49-562-75-957-97-650-18-06055-19-250-32-856-49-562-75-9

200-536-7212-072-2200-276-4200-549-8200-359-5200-015-4

Presenceofexogenousmetabolicactivation

HPRT3-MethylcholanthreneN-Nitrosodimethylamine7,12-Dimethylbenzanthracene

TK(smallandlargecolonies)

Cyclophosphamide

CyclophosphamidmonohydrateBenzo[a]pyrene3-Methylcholanthrene

200-028-5200-276-5200-549-8

XPRT

N-Nitrosodimethylamine(forhighlevelsofS-9)Benzo[a]pyrene

50-32-8200-028-5

Otherappropriatepositivecontrolreferencesubstancesmaybeused,e.g.ifalaboratoryhasahistoricaldatabaseon5-bromo2¡-deoxyuridine(CASNo59-14-3,EinecsNo200-415-9),thisreferencesubstancecouldbeusedaswell.Theuseofchemicalclass-relatedpositivecontrolchemicalsshouldbeconsidered,whenavailable.

Negativecontrols,consistingofsolventorvehiclealoneinthetreatmentmedium,andtreatedinthesamewayasthetreatmentgroups,shouldbeincluded.Inaddition,untreatedcontrolsshouldalsobeusedunlesstherearehistoricalcontroldatademonstratingthatnodeleteriousormutageniceffectsareinducedbythechosensolvent.

1.4.3.Procedure

1.4.3.1.Treatmentwiththetestsubstance

Proliferatingcellsshouldbeexposedtothetestsubstancebothwithandwithoutmetabolicactivation.Exposureshouldbeforasuitableperiodoftime(usually3󰂗6hoursiseffective).Exposuretimemaybeextendedoveronceormorecellcycles.

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Eitherduplicateorsingletreatedculturesmaybeusedateachconcentrationtested.Whensingleculturesareused,thenumberofconcentrationsshouldbeincreasedtoensureanadequatenumberofculturesforanalysis(e.g.atleasteightanalysableconcentrations).Duplicatenegative(solvent)controlculturesshouldbeused.

Gaseousorvolatilesubstancesshouldbetestedbyappropriatemethods,suchasinsealedculturevessels(21)(22).

1.4.3.2.

Measurementofsurvival,viabilityandmutantfrequency

Attheendoftheexposureperiod,cellsarewashedandculturedtodeterminesurvivalandtoallowforexpressionofthemutantphenotype.Measurementofcytotoxicitybydeterminingtherelativecloningefficiency(survival)orrelativetotalgrowthoftheculturesisusuallyinitiatedafterthetreatmentperiod.Eachlocushasadefinedminimumtimerequirementtoallownearoptimalphenotypicexpressionofnewlyinducedmutants(HPRTandXPRTrequireatleast6-8days,andTKatleasttwodays).Cellsaregrowninmediumwithandwithoutselectiveagent(s)fordeterminationofnumbersofmutantsandcloningefficiency,respectively.Themeasurementofviability(usedtocalculatemutantfrequency)isinitiatedattheendoftheexpressiontimebyplatinginnon-selectivemedium.

IfthetestsubstanceispositiveintheL5178YTK+/¯test,colonysizingshouldbeperformedonatleastoneofthetestcultures(thehighestpositiveconcentration)andonthenegativeandpositivecontrols.IfthetestsubstanceisnegativeintheL5178YTK+/¯test,colonysizingshouldbeperformedonthenegativeandpositivecontrols.InstudiesusingTK6TK+/¯,colonysizingmayalsobeperformed.

2.DATA

2.1.TREATMENTOFRESULTS

Datashouldincludecytotoxicityandviabilitydetermination,colonycountsandmutantfrequenciesforthetreatedandcontrolcultures.InthecaseofapositiveresponseintheL5178YTK+/¯test,coloniesarescoredusingthecriteriaofsmallandlargecoloniesonatleastoneconcentrationofthetestsubstance(highestpositiveconcentration)andonthenegativeandpositivecontrol.Themolecularandcytogeneticnatureofbothlargeandsmallcolonymutantshasbeenexploredindetail(23)(24).IntheTK+/¯test,coloniesarescoredusingthecriteriaofnormalgrowth(large)andslowgrowth(small)colonies(25).Mutantcellsthathavesufferedthemostextensivegeneticdamagehaveprolongeddoublingtimesandthusformsmallcolonies.Thisdamagetypicallyrangesinscalefromthelossesoftheentiregenetokaryotypicallyvisiblechromosomeaberrations.Theinductionofsmallcolonymutantshasbeenassociatedwithchemicalsthatinducegrosschromosomeaberrations(26).Lessseriouslyaffectedmutantcellsgrowatratessimilartotheparentalcellsandformlargecolonies.

Survival(relativecloningefficiencies)orrelativetotalgrowthshouldbegiven.Mutantfrequencyshouldbeexpressedasnumberofmutantcellspernumberofsurvivingcells.

Individualculturedatashouldbeprovided.Additionally,alldatashouldbesummarisedintabularform.Thereisnorequirementforverificationofaclearpositiveresponse.Equivocalresultsshouldbeclarifiedbyfurthertestingpreferablyusingmodificationofexperimentalconditions.Negativeresultsneedtobeconfirmedonacase-by-casebasis.Inthosecaseswhereconfirmationofnegativeresultsisnotconsiderednecessary,justificationshouldbeprovided.Modificationofstudyparameterstoextendtherangeofconditionsassessedshouldbeconsideredinfollow-upexperimentsforeitherequivocalornegativeresults.Studyparametersthatmightbemodifiedincludetheconcentrationspacingandthemetabolicactivationconditions.

2.2.EVALUATIONANDINTERPRETATIONOFRESULTS

Thereareseveralcriteriafordeterminingapositiveresult,suchasaconcentration-relatedincreaseorareproducibleincreaseinmutantfrequency.Biologicalrelevanceoftheresultsshouldbeconsideredfirst.Statisticalmethodsmaybeusedasanaidinevaluatingthetestresults.Statisticalsignificanceshouldnotbetheonlydeterminingfactorforapositiveresponse.

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Atestsubstanceforwhichtheresultsdonotmeettheabovecriteriaisconsiderednon-mutagenicinthissystem.

Althoughmoststudieswillgiveclearlypositiveornegativeresults,inrarecasesthedatasetwillprecludemakingadefinitejudgementabouttheactivityofthetestsubstance.Resultsmayremainequivocalorquestionableregardlessofthenumberoftimestheexperimentisrepeated.

Positiveresultsfromtheinvitromammaliancellgenemutationtestindicatethatthetestsubstanceinducesgenemutationsintheculturedmammaliancellsused.Apositiveconcentration-responsethatisreproducibleismostmeaningful.Negativeresultsindicatethat,underthetestconditions,thetestsubstancedoesnotinducegenemutationsintheculturedmammaliancellsused.

3.REPORTINGTESTREPORT

Thetestreportmustincludethefollowinginformation:Solvent/vehicle:

󰂗justificationforchoiceofvehicle/solvent,

󰂗solubilityandstabilityofthetestsubstanceinsolvent/vehicle,ifknown.Cells:

󰂗typeandsourceofcells,󰂗numberofcellcultures,

󰂗numberofcellpassages,ifapplicable,

󰂗methodsformaintenanceofcellculture,ifapplicable,󰂗absenceofmycoplasma.Testconditions:

󰂗rationaleforselectionofconcentrationsandnumberofculturesincluding,e.g.cytotoxicitydataand

solubilitylimitations,ifavailable,󰂗compositionofmedia,CO2concentration,󰂗concentrationoftestsubstance,

󰂗volumeofvehicleandtestsubstanceadded,󰂗incubationtemperature,󰂗incubationtime,󰂗durationoftreatment,󰂗celldensityduringtreatment,

󰂗typeandcompositionofmetabolicactivationsystem,includingacceptabilitycriteria,󰂗positiveandnegativecontrols,

󰂗lengthofexpressionperiod(includingnumberofcellsseeded,andsubculturesandfeedingschedules,if

appropriate),󰂗selectiveagents,

󰂗criteriaforconsideringtestsaspositive,negativeorequivocal,

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󰂗methodsusedtoenumeratenumbersofviableandmutantcells,

󰂗definitionofcoloniesofwhichsizeandtypeareconsidered(includingcriteriafor󰂓small󰂔and

󰂓large󰂔colonies,asappropriate).Results:

󰂗signsoftoxicity,󰂗signsofprecipitation,

󰂗dataonpHandosmolalityduringtheexposuretothetestsubstance,ifdetermined,󰂗colonysizeifscoredforatleastnegativeandpositivecontrols,

󰂗laboratory'sadequacytodetectsmallcolonymutantswiththeL5178YTK+/¯systemwhereappropriate,󰂗dose-responserelationship,wherepossible,󰂗statisticalanalyses,ifany,

󰂗concurrentnegative(solvent/vehicle)andpositivecontroldata,

󰂗historicalnegative(solvent/vehicle)andpositivecontroldatawithranges,meansandstandarddeviations,󰂗mutantfrequency.Discussionofresults.Conclusions.

4.REFERENCES

(1)Moore,M.M.,DeMarini,D.M.,DeSerres,F.J.andTindal,K.R.(eds.)(1987),BanburyReport28;

MammalianCellMutagenesis,ColdSpringHarborLaboratory,NewYork.(2)Chu,E.H.Y.andMallingH.V.(1968),MammalianCellGenetics.II.ChemicalInductionofSpecific

LocusMutationsinChineseHamsterCellsInVitro,Proc.Natl.Acad.Sci.USA,61,pp.1306󰂗1312.(3)Liber,H.L.andThilly,W.G.(1982),MutationAssayattheThymidineKinaseLocusinDiploidHuman

Lymphoblasts,MutationRes.94,pp.467󰂗485.(4)Moore,M.M.,Harington-Brock,K.,Doerr,C.L.andDearfield,K.L.(19),DifferentialMutant

QuantitationattheMouseLymphomaTKandCHOHGPRTLoci,Mutagenesis,4,pp.394󰂗403.(5)Aaron,C.S.,andStankowski,Jr.L.F.,(19),ComparisonoftheAS52/XPRTandtheCHO/HPRT

Assays:EvaluationofSixDrugCandidates,MutationRes.223,pp.121󰂗128.(6)Aaron,C.S.,Bolcsfoldi,G.,Glatt,H.R.,Moore,M.,Nishi,Y.,Stankowski,Jr.L.F.,Theiss,J.and

Thompson,E.(1994),MammalianCellGeneMutationAssaysWorkingGroupReport.ReportoftheInternationalWorkshoponStandardisationofGenotoxicityTestProcedures.MutationRes.312,pp.235󰂗239.(7)Scott,D.,Galloway,S.M.,Marshall,R.R.,Ishidate,M.,Brusick,D.,Ashby,J.andMyhr,B.C.(1991),

GenotoxicityUnderExtremeCultureConditions.AreportfromICPEMCTaskGroup9,MutationRes.257,pp.147󰂗204.(8)Clive,D.,McCuen,R.,Spector,J.F.S.,Piper,C.andMavournin,K.H.(1983),SpecificGeneMutations

inL5178YCellsinCulture.AReportoftheU.S.EnvironmentalProtectionAgencyGene-ToxProgram,MutationRes.,115,pp.225󰂗251.(9)Li,A.P.,Gupta,R.S.,Heflich,R.H.andWasson,J.S.(1988),AReviewandAnalysisoftheChinese

HamsterOvary/HypoxanthineGuaninePhosphoribosylTransferaseSystemtoDeterminetheMutagenicityofChemicalAgents:AReportofPhaseIIIoftheU.S.EnvironmentalProtectionsAgencyGene-ToxProgram,MutationRes.,196,pp.17󰂗36.

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(10)Li,A.P.,Carver,J.H.,Choy,W.N.,Hsie,A.W.,Gupta,R.S.,Loveday,K.S.,ÓNeill,J.P.,Riddle,J.C.,

Stankowski,L.F.Jr.andYang,L.L.(1987),AGuideforthePerformanceoftheChineseHamsterOvaryCell/Hypoxanthine-GuaninePhosphoribosylTransferaseGeneMutationAssay,MutationRes.,1,pp.135󰂗141.(11)Liber,H.L.,Yandell,D.W.andLittle,J.B.(19),AComparisonofMutationInductionattheTKand

HPRTLociinHumanLymphoblastoidCells:QuantitativeDifferencesareDuetoanAdditionalClassofMutationsattheAutosomalTKLocus,MutationRes.,216,pp.9󰂗17.(12)Stankowski,L.F.Jr.,Tindal,,K.R.,andHsie,A.W.(1986),QuantitativeandMolecularAnalysesof

EthylMethanosulphonate󰂗andICR191-InducedMolecularAnalysesofEthylMethanosulphonate󰂗andICR191InducedMutationinAS52Cells,MutationRes.,160,pp.133󰂗147.(13)Turner,N.T.,Batson,A.G.andClive,D.(1984),ProcedurefortheL5178Y/TK+/¯󰂗TK+/¯Mouse

LymphomaCellMutagenicityAssay,in:Kilbey,B.J.etal(eds.)HandbookofMutagenicityTestProcedures,ElsevierSciencePublishers,NewYork,pp.239󰂗268.(14)Arlett,C.F.,Smith,D.M.,Clarke,G.M.,Green,M.H.L.,Cole,J.,McGregor,D.B.andAsquithS.C.

(19),MammalianCellGeneMutationAssaysBaseduponColonyFormation,in:StatisticalEvaluationofMutagenicityTestData,Kirkland,D.J.,ed.,CambirdgeUniversityPress,pp.66󰂗101.(15)Abbondandolo,A.,Bonatti,S.,Corti,G.,Fiorio,R.,Loprieno,N.andMazzaccoro,A.(1977),Induction

of6-Thioguanine-ResistantMutantsinV79ChineseHamsterCellsbyMouse-LiverMicrosome-ActivatedDimethylnitrosamine,MutationRes.46,pp.365󰂗373.(16)Ames,B.N.,McCann,J.andYamasaki,E.(1975),MethodsforDetectingCarcinogensandMutagens

withtheSalmonella/Mammalian-MicrosomeMutagenicityTest,MutationRes.31,pp.347󰂗3.(17)Clive,D.,Johnson,K.O.,Spector,J.F.S.,BatsonA.G.andBrownM.M.M.(1979),Validationand

CharacterisationoftheL5178Y/TK+/¯󰂗MouseLymphomaMutagenAssaySystem,Mutat.Res.59,pp.61󰂗108.(18)Maron,D.M.andAmes,B.N.(1983),RevisedMethodsfortheSalmonellaMutagenicityTest,Mutation

Res.113,pp.173󰂗215.(19)Elliott,B.M.,Combes,R.D.,Elcombe,C.R.,Gatehouse,D.G.,Gibson,G.G.,Mackay,J.M.andWolf,

R.C.(1992),AlternativestoAroclor1254-InducedS9in:InVitroGenotoxicityAssays,Mutagenesis7,pp.175󰂗177.(20)Matsushima,T.,Sawamura,M.,Hara,K.andSugimura,T.(1976),ASafeSubstituteforPolycholrinated

BiphenylsasanInducerofMetabolicActivationSystems,in:InVitroMetabolicActivationinMutagenesisTesting,deSerres,F.J.,Fouts,J.R.,Bend,J.R.andPhilpot,R.M.(eds),Elsevier,North-Holland,pp.85󰂗88.(21)Krahn,D.F.,Barsky,F.C.andMcCooey,K.T.(1982),CHO/HGPRTMutationAssay:Evaluationof

GasesandVolatileLiquids,in:Ticc,R.R.,Costa,D.L.,Schaich,K.M.(eds),GenotoxicEffectsofAirborneAgents,NewYork,Plenum,pp.91󰂗103.(22)Zamora,P.O.,Benson,J.M.,Li,A.P.andBrooks,A.L.(1983),EvaluationofanExposureSystem

UsingCellsGrownonCollagenGelsforDetectingHighlyVolatileMutagensintheCHO/HGPRTMutationAssay,EnvironmentalMutagenesis,5,pp.795󰂗801.(23)Applegate,M.L.,Moore,M.M.,Broder,C.B.,Burrell,A.andHozier,J.C.(1990),MolecularDissection

ofMutationsattheHeterozygousThymidineKinaseLocusinMouseLymphomaCells,Proc.Natl.Acad.Sci.USA,87,pp.51󰂗55.(24)Moore,M.M.,Clive,D.Hozier,J.C.,Howard,B.E.,Batson,A.G.,Turner,N.T.andSawyer,J.(1985),

AnalysisofTrifluoronthymidine,Resistant(TFT+)MutantsofL5178Y/TK+/¯󰂗MouseLymphomaCells,MutationRes.151,pp.161󰂗174.(25)Yandell,D.W.,Dryja,T.P.andLittle,J.B.(1990),MolecularGeneticAnalysisofRecessiveMutations

ataHeterozygousAutosomalLocusinHumanCells,MutationRes.229,pp.󰂗102.(26)Moore,M.M.andDoerr,C.L.(1990),ComparisonofChromosomeAberrationFrequencyandSmall

ColonyTK-DeficientMutantFrequencyinL5178Y/TK+/¯󰂗3.7.2CMouseLymphomaCells,Mutagenesis,5,pp.609󰂗614.󰂒

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ANNEX4F

󰂑B.23.MAMMALIANSPERMATOGONIALCHROMOSOMEABERRATIONTEST

1.METHOD

ThismethodisareplicateoftheOECDTG483,MammalianSpermatogonialChromosomeAberrationTest(1997).

1.1.INTRODUCTION

Thepurposeoftheinvivomammalianspermatogonialchromosomeaberrationtestistoidentifythosesubstancesthatcausestructuralchromosomeaberrationsinmammalianspermatogonialcells(1)(2)(3)(4)(5).Structuralaberrationsmaybeoftwotypes,chromosomeorchromatid.Withthemajorityofchemicalmutagens,inducedaberrationsareofthechromatidtype,butchromosome-typeaberrationsalsooccur.Thismethodisnotdesignedtomeasurenumericalaberrationsandisnotroutinelyusedforthatpurpose.Chromosomemutationsandrelatedeventsarethecauseofmanyhumangeneticdiseases.

Thistestmeasureschromosomeeventsinspermatogonialgermcellsandis,therefore,expectedtobepredictiveofinductionofinheritablemutationsingermcells.

Rodentsareroutinelyusedinthistest.Thisinvivocytogenetictestdetectschromosomeaberrationsinspermatogonialmitoses.Othertargetcellsarenotthesubjectofthismethod.

Todetectchromatid-typeaberrationsinspermatogonialcells,thefirstmitoticcelldivisionfollowingtreatmentshouldbeexaminedbeforetheselesionsarelostinsubsequentcelldivisions.Additionalinformationfromtreatedspermatogonialstemcellscanbeobtainedbymeioticchromosomeanaylsisforchromosome-typeaberrationsatdiakinesis-metaphaseIwhenthetreatedcellsbecomespermatocytes.Thisinvivotestisdesignedtoinvestigatewhethersomaticcellmutagensarealsoactiveingermcells.Inaddition,thespermatogonialtestisrelevanttoassessingmutagenicityhazardinthatitallowsconsiderationoffactorsofinvivometabolism,pharmacokineticsandDNArepairprocesses.

Anumberofgenerationsofspermatogoniaarepresentinthetestiswithaspectrumofsensitivitytochemicaltreatment.Thus,theaberrationsdetectedrepresentanaggregateresponseoftreatedspermatogonialcellpopulations,withthemorenumerousdifferentiatedspermatogonialcellspredominating.Dependingontheirpositionwithinthetestis,differentgenerationsofspermatogoniamayormaynotbeexposedtothegeneralcirculation,becauseofthephysicalandphysiologicalSertolicellbarrierandtheblood-testisbarrier.

Ifthereisevidencethatthetestsubstance,orareactivemetabolite,willnotreachthetargettissue,itisnotappropriatetousethistest.SeealsoGeneralIntroductionPartB.

1.2.DEFINITIONS

Chromatid-typeaberration:structuralchromosomedamageexpressedasbreakageofsinglechromatidsorbreakageandreunionbetweenchromatids.

Chromosome-typeaberration:structuralchromosomedamageexpressedasbreakage,orbreakageandreunion,ofbothchromatidsatanidenticalsite.

Gap:anachromaticlesionsmallerthanthewidthofonechromatid,andwithminimummisalignmentofthechromatids.

Numericalaberration:achangeinthenumberofchromosomesfromthenormalnumbercharacteristicoftheanimalsutilised.

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Polyploidy:amultipleofthehaploidchromosomenumber(n)otherthanthediploidnumber(i.e.3n,4nandsoon).

Structuralaberration:achangeinchromosomestructuredetectablebymicroscopicexaminationofthemetaphasestageofcelldivision,observedasdeletions,intrachangesorinterchanges.

1.3.PRINCIPLEOFTHETESTMETHOD

Animalsareexposedtothetestsubstancebyanappropriaterouteofexposureandaresacrificedatappropriatetimesaftertreatment.Priortosacrifice,animalsaretreatedwithametaphase-arrestingsubstance(e.g.colchicineorColcemid®).Chromosomepreparationsarethenmadefromgermcellsandstained,andmetaphasecellsareanalysedforchromosomeaberrations.

1.4.1.4.1.1.4.1.1.

DESCRIPTIONOFTHETESTMETHODPreparations

Selectionofanimalspecies

MaleChinesehamstersandmicearecommonlyused.However,malesofotherappropriatemammalianspeciesmaybeused.Commonlyusedlaboratorystrainsofyounghealthyadultanimalsshouldbeemployed.Atthecommencementofthestudytheweightvariationofanimalsshouldbeminimalandnotexceed±20%ofthemeanweight.

1.4.1.2.Housingandfeedingconditions

GeneralconditionsreferredintheGeneralIntroductiontoPartBareappliedalthoughtheaimforhumidityshouldbe50󰂗60%.

1.4.1.3.Preparationoftheanimals

Healthyyoungadultmalesarerandomlyassignedtothecontrolandtreatmentgroups.Cagesshouldbearrangedinsuchawaythatpossibleeffectsduetocageplacementareminimised.Theanimalsareidentifieduniquely.Theanimalsareacclimatedtothelaboratoryconditionsforatleastfivedayspriortothestartofthestudy.

1.4.1.4.Preparationofdoses

Solidtestsubstanceshouldbedissolvedorsuspendedinappropriatesolventsorvehiclesanddiluted,ifappropriate,priortodosingoftheanimals.Liquidtestsubstancesmaybedoseddirectlyordilutedpriortodosing.Freshpreparationsofthetestsubstanceshouldbeemployedunlessstabilitydatademonstratetheacceptabilityofstorage.

1.4.2.1.4.2.1.

TestconditionsSolvent/vehicle

Thesolvent/vehicleshouldnotproducetoxiceffectsatthedoselevelsusedandshouldnotbesuspectedofchemicalreactionwiththetestsubstance.Ifotherthanwell-knownsolvents/vehiclesareused,theirinclusionshouldbesupportedbydataindicatingtheircompatibility.Itisrecommendedthatwhereverpossible,theuseofanaqueoussolvent/vehicleshouldbeconsideredfirst.

1.4.2.2.Controls

Concurrentpositiveandnegative(solvent/vehicle)controlsshouldbeincludedineachtest.Exceptfortreatmentwiththetestsubstance,animalsinthecontrolgroupsshouldbehandledinanidenticalmannertoanimalsinthetreatedgroups.

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Positivecontrolsshouldproducestructuralchromosomeaberrationsinvivoinspermatogonialcellswhenadministeredatexposurelevelsexpectedtogiveadetectableincreaseoverbackground.

Positivecontroldosesshouldbechosensothattheeffectsareclearbutdonotimmediatelyrevealtheidentityofthecodedslidestothereader.Itisacceptablethatthepositivecontrolbeadministeredbyaroutedifferentfromthetestsubstanceandsampledatonlyasingletime.Inaddition,theuseofchemicalclass-relatedpositivecontrolchemicalsmaybeconsidered,whenavailable.Examplesofpositivecontrolsubstancesinclude:

Substance

CASNo

EinecsNo

cyclophosphamide

cyclophosphamidemonohydratecyclohexylaminemitomycinCmonomericacrylamidetriethylenemelamine

50-18-06055-19-2108-91-850-07-779-06-151-18-3

200-015-4

203-629-0200-008-6201-173-7200-083-5

Negativecontrols,treatedwithsolventorvehiclealone,andotherwisetreatedinthesamewayasthetreatmentgroups,shouldbeincludedforeverysamplingtime,unlessacceptableinter-animalvariabilityandfrequencyofcellswithchromosomeaberrationsaredemonstratedbyhistoricalcontroldata.Inaddition,untreatedcontrolsshouldalsobeusedunlesstherearehistoricalorpublishedcontroldatademonstratingthatnodeleteriousormutageniceffectsareinducedbythechosensolvent/vehicle.

1.5.PROCEDURE

1.5.1.Numberofanimals

Eachtreatedandcontrolgroupmustincludeatleastfiveanalysablemales.

1.5.2.Treatmentschedule

Testsubstancesarepreferablyadministeredonceortwice(i.e.asasingletreatmentorastwotreatments).Testsubstancesmayalsobeadministeredasasplitdose,i.e.twotreatmentsonthesamedayseparatedbynomorethanafewhours,tofacilitateadministeringalargevolumeofmaterial.Otherdoseregimensshouldbescientificallyjustified.

Inthehighestdosegrouptwosamplingtimesaftertreatmentareused.Sincecellcyclekineticscanbeinfluencedbythetestsubstance,oneearlyandonelatesamplingtimeareusedaround24and48hoursaftertreatment.Fordosesotherthanthehighestdose,asamplingtimeof24hoursor1,5cellcyclelengthaftertreatmentshouldbetaken,unlessanothersamplingtimeisknowntobemoreappropriatefordetectionofeffects(6).

Inaddition,othersamplingtimesmaybeused.Forexampleinthecaseofchemicalswhichmayinducechromosomelagging,ormayexertS-independenteffects,earliersamplingtimesmaybeappropriate(1).Theappropriatenessofarepeatedtreatmentscheduleneedstobeidentifiedonacase-by-casebasis.Followingarepeatedtreatmentscheduletheanimalsshouldthenbesacrificed24hours(1,5cellcyclelength)afterthelasttreatment.Additionalsamplingtimesmaybeusedwhereappropriate.

Priortosacrifice,animalsareinjectedintraperitoneallywithanappropriatedoseofametaphasearrestingsubstance(e.g.Colcemid®orcolchocine).Animalsaresampledatanappropriateintervalthereafter.Formicethisintervalisapproximately3󰂗5hours,forChinesehamstersthisintervalisapproximately4󰂗5hours.

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1.5.3.

ENDoselevels

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Ifarangefindingstudyisperformedbecausetherearenosuitabledataavailable,itshouldbeperformedinthesamelaboratory,usingthesamespecies,strainandtreatmentregimentobeusedinthemainstudy(7).Ifthereistoxicity,threedoselevelsareusedforthefirstsamplingtime.Thesedoselevelsshouldcoverarangefromthemaximumtolittleornotoxicity.Atthelatersamplingtimeonlythehighestdoseneedstobeused.Thehighestdoseisdefinedasthedose-producingsignsoftoxicitysuchthathigherdoselevels,basedonthesamedosingregimen,wouldbeexpectedtoproducelethality.

Substanceswithspecificbiologicalactivitiesatlownon-toxicdoses(suchashormonesandmitogens)maybeexceptionstothedose-settingcriteriaandshouldbeevaluatedonacase-by-casebasis.Thehighestdosemayalsobedefinedasadosethatproducessomeindicationoftoxicityinthespermatogonialcells(e.g.areductionintheratioofspermatogonialmitosestofirstandsecondmeioticmetaphases;thisreductionshouldnotexceed50%).

1.5.4.

Limittest

Ifatestatonedoselevelofatleast2000mg/kgbodyweight/dayusingasingletreatment,orastwotreatmentsonthesameday,producesnoobservabletoxiceffects,andifgenotoxicitywouldnotbeexpectedbasedupondatafromstructurallyrelatedsubstances,thenafullstudyusingthreedoselevelsmaynotbeconsiderednecessary.Expectedhumanexposuremayindicatetheneedforahigherdoseleveltobeusedinthelimittest.

1.5.5.

Administrationofdoses

Thetestsubstanceisusuallyadministeredbygavageusingastomachtubeorasuitableintubationcannula,orbyintraperitonealinjection.Otherroutesofexposuremaybeacceptablewheretheycanbejustified.Themaximumvolumeofliquidthatcanbeadministeredbygavageorinjectionatonetimedependsonthesizeofthetestanimal.Thevolumeshouldnotexceed2ml/100gbodyweight.Theuseofvolumeshigherthanthesemustbejustified.Exceptforirritatingorcorrosivesubstances,whichwillnormallyrevealexacerbatedeffectswithhigherconcentrations,variabilityintestvolumeshouldbeminimisedbyadjustingtheconcentrationtoensureaconstantvolumeatalldoselevels.

1.5.6.

Chromosomepreparation

Immediatelyaftersacrifice,cellsuspensionsareobtainedfromoneorbothtestes,exposedtohypotonicsolutionandfixed.Thecellsarethenspreadonslidesandstained.

1.5.7.

Analysis

Foreachanimalatleast100well-spreadmetaphaseshouldbeanalysed(i.e.aminimumof500metaphasespergroup).Thisnumbercouldbereducedwhenhighnumbersofaberrationsareobserved.Allslides,includingthoseofpositiveandnegativecontrols,shouldbeindependentlycodedbeforemicroscopicanalysis.Sincefixationproceduresoftenresultinthebreakageofaproportionofmetaphaseswithlossofchromosomes,thecellsscoredshouldcontainanumberofcentromeresequaltothenumber2n±2.

2.DATA

2.1.TREATMENTOFRESULTS

Individualanimaldatashouldbepresentedinatabularform.Theexperimentalunitistheanimal.Foreachindividualanimalthenumberofcellswithstructuralchromosomeaberrationsandthenumberofchromosomeaberrationspercellshouldbeevaluated.Differenttypesofstructuralchromosomeaberrationsshouldbelistedwiththeirnumbersandfrequenciesfortreatedandcontrolgroups.Gapsarerecordedseparatelyandreportedbutgenerallynotincludedinthetotalaberrationfrequency.

Ifmitosisaswellasmeiosisisobserved,theratioofspermatogonialmitosestofirstandsecondmeioticmetaphasesshouldbedeterminedasameasureofcytotoxicityforalltreatedandnegativecontrolanimalsinatotalsampleof100dividingcellsperanimaltoestablishapossiblecytotoxiceffect.Ifonlymitosisisobserved,themitosisindexshouldbedeterminedinatleast1000cellsforeachanimal.

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2.2.

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EVALUATIONANDINTERPRETATIONOFRESULTS

Thereareseveralcriteriafordeterminingapositiveresult,suchasadose-relatedincreaseintherelativenumberofcellswithchromosomeaberrationsoraclearincreaseinthenumberofcellswithaberrationsinasingledoseatasinglesamplingtime.Biologicalrelevanceoftheresultsshouldbeconsideredfirst.Statisticalmethodsmaybeusedasanaidinevaluatingthetestresults(8).Statisticalsignificanceshouldnotbetheonlydeterminingfactorforapositiveresponse.Equivocalresultsshouldbeclarifiedbyfurthertestingpreferablyusingamodificationofexperimentalconditions.

Atestsubstanceforwhichtheresultsdonotmeettheabovecriteriaisconsiderednon-mutagenicinthistest.Althoughmostexperimentswillgiveclearlypositiveornegativeresults,inrarecasesthedatasetwillprecludemakingadefinitejudgementabouttheactivityofthetestsubstance.Resultsmayremainequivocalorquestionableregardlessofthenumberoftimestheexperimentisrepeated.

Positiveresultsfromtheinvivospermatogonialchromosomeaberrationtestindicatethatthetestsubstanceinducesstructuralchromosomeaberrationsinthegermcellsofthespeciestested.Negativeresultsindicatethat,underthetestconditions,thetestsubstancedoesnotinducechromosomeaberrationsinthegermcellsofthespeciestested.

Thelikelihoodthatthetestsubstanceoritsmetabolitesreachthetargettissueshouldbediscussed.

3.REPORTINGTESTREPORT

Thetestreportmustincludethefollowinginformation:Solvent/vehicle:

󰂗justificationforchoiceofvehicle,

󰂗solubilityandstabilityofthetestsubstanceinsolvent/vehicle,ifknown.Testanimals:

󰂗species/strainused,󰂗numberandageofanimals,󰂗source,housingconditions,diet,etc.,

󰂗individualweightoftheanimalsatthestartofthetest,includingbodyweightrange,meanandstandard

deviationforeachgroup.Testconditions:

󰂗datafromrangefindingstudy,ifconcluded,󰂗rationalefordoselevelselection,󰂗rationaleforrouteofadministration,󰂗detailsoftestsubstancepreparation,

󰂗detailsoftheadministrationofthetestsubstance,󰂗rationaleforsacrificetimes,

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󰂗conversionfromdiet/drinkingwatertestsubstanceconcentration(ppm)totheactualdose(mg/kgbody

weight/day),ifapplicable,󰂗detailsoffoodandwaterquality,

󰂗detaileddescriptionoftreatmentandsamplingschedules,󰂗methodsformeasurementoftoxicity,

󰂗identityofmetaphasearrestingsubstance,itsconcentrationanddurationoftreatment,󰂗methodsofslidepreparation,󰂗criteriaforscoringaberrations,󰂗numberofcellsanalysedperanimal,

󰂗criteriaforconsideringstudiesaspositive,negativeorequivocal.Results:

󰂗signsoftoxicity,󰂗mitoticindex,

󰂗ratioofspermatogonialmitosescellstofirstandsecondmeioticmetaphases,󰂗typeandnumberofaberrations,givenseparatelyforeachanimal,󰂗totalnumberofaberrationspergroup,󰂗numberofcellswithaberrationspergroup,󰂗dose-responserelationship,ifpossible,󰂗statisticalanalyses,ifany,󰂗concurrentnegativecontroldata,

󰂗historicalnegativecontroldatawithranges,meansandstandarddeviations,󰂗concurrentpositivecontroldata,󰂗changesinploidy,ifseen.Discussionofresults.Conclusions.

4.REFERENCES

(1)Adler,I.D.,(1986),ClastogenicPotentialinMouseSpermatogoniaofChemicalMutagensRelatedtotheir

Cell-CycleSpecifications,in:GeneticToxicologyofEnvironmentalChemicals,PartB:GeneticEffectsandAppliedMutagenesis,Ramel,C.,Lambert,B.andMagnusson,J.(eds)Liss,NewYork,pp.477-484.(2)Adler,I.D.,(1984),CytogenetictestsinMammals,in:MutagenicityTesting:aPracticalApproach,(ed.)S.

VenittandJ.M.Parry,IRLPress,Oxford,WashingtonDC,pp.275-306.(3)Evans,E.P.,Breckon,G.andFord,C.E.(19),AnAir-dryingMethodforMeioticPreparationsfrom

MammalianTestes,CytogeneticsandCellGenetics,3,pp.2-294.

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(4)Richold,M.,Ashby,J.,Chandley,A.,Gatehouse,D.G.andHenderson,L.(1990),InVivoCytogenetic

Assays,in:D.J.Kirkland(ed.),BasicMutagenicityTests,UKEMSRecommendedProcedures.UKEMSSubcommitteeonGuidelinesforMutagenicityTesting.Report.PartIrevised,CambridgeUniversityPress,Cambridge,NewYork,PortChester,Melbourne,Sydney,pp.115-141.(5)Yamamoto,K.andKikuchi,Y.(1978),ANewMethodforPreparationofMammalianSpermatogonial

Chromosomes,MutationRes.,52,pp.207-209.(6)Adler,I.D.,ShelbyM.D.,Bootman,J.,Favor,J.,Generoso,W.,Pacchierotti,F.,Shibuya,T.andTanaka

N.(1994),InternationalWorkshoponStandardisationofGenotoxicityTestProcedures.SummaryReportoftheWorkingGrouponMammalianGermCellTests,MutationRes.,312,pp.313-318.(7)Fielder,R.J.,Allen,J.A.,Boobis,A.R.,Botham,P.A.,Doe,J.,Esdaile,D.J.,Gatehouse,D.G.,

Hodson-Walker,G.,Morton,D.B.,Kirkland,D.J.andRichold,M.(1992),ReportofBritishToxicologySociety/UKEnvironmentalMutagenSocietyWorkingGroup:DosesettinginInVivoMutagenicityAssays,Mutagenesis,7,pp.313-319.(8)Lovell,D.P.,Anderson,D.,Albanese,R.,Amphlett,G.E.,Clarc,G.,Ferguson,R.,Richold,M.,Papworth,

D.G.andSavageJ.R.K.(19),StatisticalAnalysisofInVivoCytogeneticAssays,in:D.J.Kirkland(ed.),StatisticalEvaluationofMutagenicityTestData.UKEMSSubcommitteeonGuidelinesforMutagenicityTesting,report,PartIII.CambridgeUniversityPress,Cambridge,NewYork,PortChester,Melbourne,Sydney,pp.184-232.󰂒

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ANNEX4G

󰂑B.39.UNSCHEDULEDDNASYNTHESIS(UDS)TESTWITHMAMMALIANLIVERCELLSINVIVO

1.METHOD

ThismethodisareplicateoftheOECDTG486,UnscheduledDNASynthesis(UDS)TestwithMammalianLiverCellsInVivo(1997).

1.1.INTRODUCTION

ThepurposeoftheunscheduledDNAsynthesis(UDS)testwithmammalianlivercellsinvivoistoidentifytestsubstancesthatinduceDNArepairinlivercellsoftreatedanimals(1),(2),(3),(4).

Thisinvivotestprovidesamethodforinvestigatinggenotoxiceffectsofchemicalsintheliver.TheendpointmeasuredisindicativeofDNAdamageandsubsequentrepairinlivercells.Theliverisusuallythemajorsiteofmetabolismofabsorbedcompounds.ItisthusanappropriatesitetomeasureDNAdamageinvivo.Ifthereisevidencethatthetestsubstancewillnotreachthetargettissue,itisnotappropriatetousethistest.TheendpointofunscheduledDNAsynthesis(UDS)ismeasuredbydeterminingtheuptakeoflabellednucleosidesincellsthatarenotundergoingscheduled(S-phase)DNAsynthesis.Themostwidelyusedtechniqueisthedeterminationoftheuptakeoftritium-labelledthymidine(3H-TdR)byautoradiography.RatliversarepreferablyusedforinvivoUDStests.Tissuesotherthantheliversmaybeused,butarenotthesubjectofthismethod.

ThedetectionofaUDSresponseisdependentonthenumberofDNAbasesexcisedandreplacedatthesiteofthedamage.Therefore,theUDStestisparticularlyvaluabletodetectsubstance-induced󰂓longpatchrepair󰂔(20to30bases).Incontrast󰂓shortpatchrepair󰂔(onetothreebases)isdetectedwithmuchlowersensitivity.Furthermore,mutageniceventsmayresultbecauseofnon-repair,misrepairormisreplicationofDNAlesions.TheextentoftheUDSresponsegivesnoindicationofthefidelityoftherepairprocess.Inaddition,itispossiblethatamutagenreactswithDNAbuttheDNAdamageisnotrepairedviaanexcisionrepairprocess.ThelackofspecificinformationonmutagenicactivityprovidedbytheUDStestiscompensatedforbythepotentialsensitivityofthisendpointbecauseitismeasuredinthewholegenome.SeealsoGeneralIntroductionPartB.

1.2.DEFINITIONS

Cellsinrepair:anetnucleargrain(NNG)higherthanapresetvalue,tobejustifiedatthelaboratoryconductingthetest.

Netnucleargrains(NNG):quantitativemeasureforUDSactivityofcellsinautoradiographicUDStests,calculatedbysubtractingtheaveragenumberofcytoplasmicgrainsinnucleus-equivalentcytoplasmicareas(CG)fromthenumberofnucleargrains(NG):NNG=NG¯CG.NNGcountsarecalculatedforindividualcellsandthenpooledforcellsinaculture,inparallelcultures,etc.

UnscheduledDNAsynthesis(UDS):DNArepairsynthesisafterexcisionandremovalofastretchofDNAcontainingaregionofdamageinducedbychemicalsubstancesorphysicalagents.

1.3.PRINCIPLEOFTHETESTMETHOD

TheUDStestwithmammalianlivercellsinvivoindicatesDNArepairsynthesisafterexcisionandremovalofastretchofDNAcontainingaregionofdamageinducedbychemicalsubstancesorphysicalagents.Thetestisusuallybasedontheincorporationof3H-TdRintotheDNAoflivercellswhichhavealowfrequencyofcellsintheS-phaseofthecellcycle.Theuptakeof3H-TdRisusuallydeterminedbyautoradiography,sincethistechniqueisnotassusceptibletointerferencefromS-phasecellsas,forexample,liquidscintillationcounting.

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1.4.1.4.1.1.4.1.1.

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DESCRIPTIONOFTHEMETHODPreparations

Selectionsofanimalspecies

Ratsarecommonlyused,althoughanyappropriatemammalianspeciesmaybeused.Commonlyusedlaboratorystrainsofyounghealthyadultanimalsshouldbeemployed.Atthecommencementofthestudytheweightvariationofanimalsshouldbeminimalandnotexceed±20%ofthemeanweightforeachsex.

1.4.1.2.Housingandfeedingconditions

GeneralconditionsreferredintheGeneralIntroductiontoPartBareappliedalthoughtheaimforhumidityshouldbe50to60%.

1.4.1.3.Preparationoftheanimals

Healthyyoungadultanimalsarerandomlyassignedtothecontrolandtreatmentgroups.Cagesshouldbearrangedinsuchawaythatpossibleeffectsduetocageplacementareminimised.Theanimalsareidentifieduniquelyandkeptintheircagesforatleastfivedayspriortothestartofthestudytoallowforacclimatisationtothelaboratoryconditions.

1.4.1.4.Testsubstance/preparation

Solidtestsubstancesshouldbedissolvedorsuspendedinappropriatesolventsorvehiclesanddiluted,ifappropriate,priortodosingoftheanimals.Liquidtestsubstancesmaybedoseddirectlyordilutedpriortodosing.Freshpreparationsofthetestsubstanceshouldbeemployedunlessstabilitydatademonstratetheacceptabilityofstorage.

1.4.2.1.4.2.1.

TestconditionsSolvent/vehicle

Thesolvent/vehicleshouldnotproducetoxiceffectsatthedoselevelsused,andshouldnotbesuspectedofchemicalreactionwiththetestsubstance.Ifotherthanwell-knownsolvents/vehiclesareused,theirinclusionshouldbesupportedwithdataindicatingtheircompatibility.Itisrecommendedthatwhereverpossible,theuseofanaqueoussolvent/vehicleshouldbeconsideredfirst.

1.4.2.2.Controls

Concurrentpositiveandnegativecontrols(solvent/vehicle)shouldbeincludedineachindependentlyperformedpartoftheexperiment.Exceptfortreatmentwiththetestsubstance,animalsinthecontrolgroupshouldbehandledinanidenticalmannertotheanimalsinthetreatedgroups.

PositivecontrolsshouldbesubstancesknowntoproduceUDSwhenadministeredatexposurelevelsexpectedtogiveadetectableincreaseoverbackground.Positivecontrolsneedingmetabolicactivationshouldbeusedatdoseselicitingamoderateresponse(4).Thedosesmaybechosensothattheeffectsareclearbutdonotimmediatelyrevealtheidentityofthecodedslidestothereader.Examplesofpositivecontrolsubstancesinclude:

Samplingtimes

Substance

CAS-No

EinecsNo

Earlysamplingtimes(2to4hours)Latesamplingtimes(12to16hours)

N-nitrosodimethylamineN-2-fluorenylacetamide(2-AAF)

62-75-953-96-3

200-249-8200-188-6

Otherappropriatepositivecontrolsubstancesmaybeused.Itisacceptablethatthepositivecontrolshouldbeadministeredbyaroutedifferentfromthetestsubstance.

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1.5.

ENPROCEDURE

OfficialJournaloftheEuropeanCommunities8.6.2000

1.5.1.Numberandsexofanimals

Anadequatenumberofanimalsshouldbeusedtotakeaccountofnaturalbiologicalvariationintestresponse.Thenumberofanimalshouldbeatleastthreeanalysableanimalspergroup.Whereasignificanthistoricaldatabasehasbeenaccumulated,onlyoneortwoanimalsarerequiredfortheconcurrentnegativeandpositivecontrolgroups.

Ifatthetimeofthestudytherearedataavailablefromstudiesinthesamespeciesandusingthesamerouteofexposurethatdemonstratethattherearenosubstantialdifferencesintoxicitybetweensexes,thentestinginasinglesex,preferablymales,willbesufficient.Wherehumanexposuretochemicalsmaybesex-specific,asforexamplewithsomepharmaceuticalagents,thetestshouldbeperformedwithanimalsoftheappropriatesex.

1.5.2.Treatmentschedule

Testsubstancesaregenerallyadministeredasasingletreatment.

1.5.3.Doselevels

Normally,atleasttwodoselevelsareused.Thehighestdoseisdefinedasthedoseproducingsignsoftoxicitysuchthathigherdoselevels,basedonthesamedosingregimen,wouldbeexpectedtoproducelethality.Ingeneral,thelowerdoseshouldbe50%to25%ofthehighdose.

Substanceswithspecificbiologicalactivitiesatlownon-toxicdoses(suchashormonesandmitogens)maybeexceptionstothedose-settingcriteriaandshouldbeevaluatedonacase-by-casebasis.Ifarangefindingstudyisperformedbecausetherearenosuitabledataavailable,itshouldbeperformedinthesamelaboratory,usingthesamespecies,strain,sex,andtreatmentregimentobeusedinthemainstudy.

Thehighestdosemayalsobedefinedasadosethatproducessomeindicationoftoxicityintheliver(e.g.pyknoticnuclei).

1.5.4.Limittest

Ifatestatonedoselevelofatleast2000mg/kgbodyweight,appliedinasingletreatment,orintwotreatmentsonthesameday,producesnoobservabletoxiceffects,andifgenotoxicitywouldnotbeexpected,basedupondatafromstructurallyrelatedsubstances,thenafullstudymaynotbenecessary.Expectedhumanexposuremayindicatetheneedforahigherdoseleveltobeusedinthelimittest.

1.5.5.Administrationofdoses

Thetestsubstanceisusuallyadministeredbygavageusingastomachtubeorasuitableintubationcannula.Otherroutesofexposuremaybeacceptablewheretheycanbejustified.However,theintraperitonealrouteisnotrecommendedasitcouldexposetheliverdirectlytothetestsubstanceratherthanviathecirculatorysystem.Themaximumvolumeofliquidthatcanbeadministeredbygavageorinjectionatonetimedependsonthesizeofthetestanimal.Thevolumeshouldnotexceed2ml/100gbodyweight.Theuseofvolumeshigherthanthesemustbejustified.Exceptforirritatingorcorrosivesubstances,whichwillnormallyrevealexacerbatedeffectswithhigherconcentrations,variabilityintestvolumeshouldbeminimisedbyadjustingtheconcentrationtoensureaconstantvolumeatalldoselevels.

1.5.6.Preparationoflivercells

Livercellsarepreparedfromtreatedanimalsnormally12to16hoursafterdosing.Anadditionalearliersamplingtime(normallytwotofourhourspost-treatment)isgenerallynecessaryunlessthereisaclearpositiveresponseat12to16hours.However,alternativesamplingtimesmaybeusedwhenjustifiedonthebasisoftoxicokineticdata.

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Short-termculturesofmammalianlivercellsareusuallyestablishedbyperfusingtheliverinsituwithcollagenaseandallowingfreshlydissociatedlivercellstoattachthemselvestoasuitablesurface.Livercellsfromnegativecontrolanimalsshouldhaveaviability(5)ofatleast50%.

1.5.7.DeterminationofUDS

Freshlyisolatedmammalianlivercellsareincubatedusuallywithmediumcontaining3H-TdRforanappropriatelengthoftime,e.g.threetoeighthours.Attheendoftheincubationperiod,mediumshouldberemovedfromthecells,whichmaythenbeincubatedwithmediumcontainingexcessunlabelledthymidinetodiminishunincorporatedradioactivity(󰂓coldchase󰂔).Thecellsarethenrinsed,fixedanddried.Formoreprolongedincubationtimes,coldchasemaynotbenecessary.Slidesaredippedinautoradiographicemulsion,exposedinthedark(e.g.refrigeratedfor7to14days),developed,stained,andexposedsilvergrainsarecounted.Twotothreeslidesarepreparedfromeachanimal.

1.5.8.Analysis

TheslidepreparationsshouldcontainsufficientcellsofnormalmorphologytopermitameaningfulassessmentofUDS.Preparationsareexaminedmicroscopicallyforsignsofovertcytoxicity(e.g.pyknosis,reducedlevelsofradiolabelling).

Slidesshouldbecodedbeforegraincounting.Normally100cellsarescoredfromeachanimalfromatleasttwoslides;thescoringoflessthan100cells/animalsshouldbejustified.GraincountsarenotscoredforS-phasenuclei,buttheproportionofS-phasecellsmayberecorded.

Theamountof3H-TdRincorporationinthenucleiandthecytoplasmofmorphologicallynormalcells,asevidencedbythedepositionofsilvergrains,shouldbedeterminedbysuitablemethods.

2.DATA

2.1.TREATMENTOFRESULTS

Individualslideandanimaldatashouldbeprovided.Additionally,alldatashouldbesummarisedintabularform.Netnucleargrain(NNG)countsshouldbecalculatedforeachcell,foreachanimalandforeachdoseandtimebysubtractingCGcountsfromNGcounts.If󰂓cellsinrepair󰂔arecounted,thecriteriafordefining󰂓cellsinrepair󰂔shouldbejustifiedandbasedonhistoricalorconcurrentnegativecontroldata.Numericalresultsmaybeevaluatedbystatisticalmethods.Ifused,statisticaltestsshouldbeselectedandjustifiedpriortoconductingthestudy.

2.2.EVALUATIONANDINTERPRETATIONOFRESULTSExamplesofcriteriaforpositive/negativeresponsesinclude:positiveornegativeor

(i)(ii)(i)(ii)

NNGvaluesaboveapresetthresholdwhichisjustifiedonthebasisoflaboratoryhistoricaldata;

NNGvaluessignificantlygreaterthanconcurrentcontrol;NNGvalueswithin/belowhistoricalcontrolthreshold;NNGvaluesnotsignificantlygreaterthanconcurrentcontrol.

Thebiologicalrelevanceofdatashouldbeconsidered:i.e.parameterssuchasinter-animalvariation,doseresponserelationshipandcytotoxicityshouldbetakenintoaccount.Statisticalmethodsmaybeusedasanaidinevaluatingthetestresults.However,statisticalsignificanceshouldnotbetheonlydeterminingfactorforapositiveresponse.

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Althoughmostexperimentswillgiveclearlypositiveornegativeresults,inrarecasesthedatasetwillprecludemakingadefinitejudgementabouttheactivityofthetestsubstance.Resultsmayremainequivocalorquestionableregardlessofthenumberoftimestheexperimentisrepeated.

ApositiveresultfromtheUDStestwithmammalianlivercellsinvivoindicatesthatatestsubstanceinducesDNAdamageinmammalianlivercellsinvivothatcanberepairedbyunscheduledDNAsynthesisinvitro.Anegativeresultindicatesthat,underthetestconditions,thetestsubstancedoesnotinduceDNAdamagethatisdetectablebythistest.

Thelikelihoodthatthetestsubstancereachesthegeneralcirculationorspecificallythetargettissue(e.g.systemictoxicity)shouldbediscussed.

3.REPORTINGTESTREPORT

Thetestreportmustincludethefollowinginformation.Solventvehicle:

󰂗justificationforchoiceofvehicle,

󰂗solubilityandstabilityofthetestsubstanceinsolvent/vehicle,ifknown.Testanimals:

󰂗species/strainused,

󰂗number,ageandsexofanimals,󰂗source,housingconditions,diet,etc.,

󰂗individualweightoftheanimalsatthestartofthetest,includingbodyweightrange,meanandstandard

deviationforeachgroup.Testconditions:

󰂗positiveandnegativevehicle/solventcontrols,󰂗datafromrange-findingstudy,ifconducted,󰂗rationalefordoselevelselection,󰂗detailsoftestsubstancepreparation,

󰂗detailsoftheadministrationofthetestsubstance,󰂗rationaleforrouteofadministration,

󰂗methodsforverifyingthattestagentreachedthegeneralcirculationortargettissue,ifapplicable,󰂗conversionfromdiet/drinkingwatertestsubstanceconcentration(ppm)totheactualdose(mg/kgbody

weight/day),ifapplicable,󰂗detailsoffoodandwaterquality,

󰂗detaileddescriptionoftreatmentandsamplingschedules,󰂗methodsformeasurementoftoxicity,󰂗methodoflivercellpreparationandculture,󰂗autoradiographictechniqueused,

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󰂗numberofslidespreparedandnumbersofcellsscored,󰂗evaluationcriteria,

󰂗criteriaforconsideringstudiesaspositive,negativeorequivocal.Results:

󰂗individualslide,animalandgroupmeanvaluesfornucleargrains,cytoplasmicgrains,andnetnuclear

grains,󰂗dose-responserelationship,ifavailable,󰂗statisticalevaluationifany,󰂗signsoftoxicity,

󰂗concurrentnegative(solvent/vehicle)andpositivecontroldata,

󰂗historicalnegative(solvent/vehicle)andpositivecontroldatawithrange,meansandstandarddeviations,󰂗numberof󰂓cellsinrepair󰂔ifdetermined,󰂗numberofS-phasecellsifdetermined,󰂗viabilityofthecells.DiscussionofresultsConclusions

4.REFERENCES

(1)Ashby,J.Lefevre,P.A.,Burlinson,B.andPenman,M.G.(1985),AnAssessmentoftheInVivoRat.

HepatocyteDNARepairAssay,MutationRes.,156,pp.1󰂗18.(2)Butterworth,B.E.,Ashby,J.,Bermudez,E.,Casciano,D.,Mirsalis,J.,Probst,G.andWilliams,G.(1987),

AProtocolandGuidefortheInVivoRatHepatocyteDNARepairAssay,MutationRes.,1,pp.123󰂗133.(3)Kennelly,J.C.,Waters,R.,Ashby,J.,Lefevre,P.A.,Burlinson,B.,Benford,D.J.,Dean,S.W.and

Mitchell,I.deG.(1993),InVivoRatLiverUDSAssay,in:KirklandD.J.andFoxM.,(eds),SupplementaryMutagenicityTests:UKEMRecommendedProcedures.UKEMSSubcommitteeonGuidelinesforMutagenicityTesting.Report.PartIIrevised,CambridgeUniversityPress,Cambridge,NewYork,PortChester,Melbourne,Sydney,pp.52󰂗77.(4)Madle,S.,Dean,S.W.,Andrac,U.,Brambilla.G.,Burlinson,B.,Doolittle,D.J.,Furihata,C.,Hertner,T.,

McQueen,C.A.andMori,H.(1993),RecommendationsforthePerformanceofUDSTestsInVitroandInVivo,MutationRes.,312,pp.263󰂗285.(5)Fautz,R.,Hussain,B.,Efstathiou,E.andHechenberger-Freudl.C.(1993),AssessmentoftheRelation

BetweentheInitialViabilityandtheAttachmentofFreshlyIsolatedRatHepatocytesUsedfortheInVivo/InVitroDNARepairAssay(UDS),MutationRes.,291,pp.21󰂗27.(6)Mirsalis,J.C.,Tyson,C.K.andButterworth,B.E.(1982),DetectionofGenotoxicCarcinogensintheIn

Vivo/InVitroHepalocyteDNARepairAssay,Environ.Mutagen,4,pp.553󰂗562.󰂒

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ANNEX5

SV:3.2.3.

Farligt

R65Farligt:kangelungskadorvidförtäring.

Flytandeämnenochberedningarsompågrundavsinlågaviskositetutgörenfaraförmänniskavidaspirationa)

Förämnenochberedningarsominnehålleralifatiska,alicykliskaocharomatiskakolvätenientotalkon-centrationav10%ellermeroch

󰂗harenflödestidmindreän30sekunder,uppmättmeden3mmutloppsbägareenligtISO2431,eller󰂗harenkinematiskviskositetlägreän7×10¯6m2/svid40°C,uppmättmedenkalibreradkapillärvis-kosimeteravglas,enligtISO3104ochISO3105,eller󰂗harenkinematiskviskositetlägreän7×10¯6m2/svid40°C,bestämdfrånrotationsviskosimetrienligt

ISO3219.Ämnenochberedningar,somuppfyllerdessakriterier,behöverdockinteklassificerasomdeharengenomsnitligytspänninghögreän33mN/mvid25°C,uppmättmedduNoüytensiometerellerenligtdetestmetodersomfinnsbeskrivnaibilagaVdelA.5.

b)Förämnenochberedningar,baseratpåpraktiskaerfarenheterfrånmänniska.

(DoesnotconcerntheESversion)(DoesnotconcerntheDAversion)(DoesnotconcerntheDEversion)(DoesnotconcerntheELversion)(DoesnotconcerntheENversion)(DoesnotconcerntheFRversion)(DoesnotconcerntheITversion)(DoesnotconcerntheNLversion)(DoesnotconcernthePTversion)(DoesnotconcerntheFIversion)

FI:

3.2.6.1Ihontulehtuminen

Seuraavavaaraaosoittavalausekamääräytyyallaesiteltävienperusteittenmukaan:R38Ärsyttääihoa

󰂗Aineetjavalmisteetaiheuttavatihonmerkittäväntulehtumisenenintäänneljäntunninaltistuksessamää-ritettynäkanillaliitteessäVmainitullaihoärsytstestillä.Tulehduskestäävähintään24tuntia.

Ihontulehdusonmerkittävää,jos:a)

punoituksenjaruvenmuodostuksentaiturvotuksenvoimakkuuttakuvaavienlukuarvojenkeskiarvolaskettunakaikistakoe-eläimistäonvähintään2;

b)taikunliitteessäVtarkoitettuatestiäontäydennettykäyttämälläkolmeakoe-eläintä,vähintäänkahden

koe-eläimenihonpunoituksenjaruvenmuodostuksentaiturvotuksenvoimakkuuttakuvaavienlukuarvojenkeskiarvoon,jokaisellekoe-eläimellelaskettunaerikseen,vähintään2.Kummassakintapauksessakeskiarvojenlasekmiseenonkäytettäväkaikkianiitälukuarvoja,jotkasaadaanarvioitaessavaikutusta24tunnin,48tunninja72tunninvälein.

8.6.2000ENOfficialJournaloftheEuropeanCommunities

Tulehdustapidetäänmyösmerkittävänä,josihontulehtuminenjatkuuainakinkahdellakoe-eläimellähavainnointiajanpäättymiseenasti.Erityisetvaikutuksetkutenesimerkiksihyperplasia,hilseileminen,värinmuutokset,halkeamat,ruvetjakarvojenlähtöonotettavahuomioon.

Tähänliittyviätietojavoidaansaadamyöseläimillätehtävistäei-akuuttisistaaltistuskokeista(katsolause-kettaR48koskevathuomautuksetjaksossa2.d).Vaikutuksiapidetäänmerkittävinä,josnevastaavatedelläkuvattujavaikutuksia.

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󰂗Aineetjavalmisteet,jotkaaiheuttavatihmisillämerkittävääihotulehdusta,kunkosketusonollutvälitön,

jatkuvataitoistuva.󰂗Orgaanisetperoksidit,paitsijosonolemassanäyttöäsiitä,ettätällaistavaikutustaeiole.Tuntoharha(󰂒paresthesia󰂒):

Pyretroiditorjunta-aineenihokosketuksenaiheuttamaatuntoharhaaihmisessäeipidetäärsytysvaikutuksena,jokaoikeuttaisiluokituksenXi;R38.S-lausekettaS24onkuitenkinsovellettavaaineisiin,joillaontällainenvaikutus.

(DoesnotconcerntheESversion)(DoesnotconcerntheDAversion)(DoesnotconcerntheDEversion)(DoesnotconcerntheELversion)(DoesnotconcerntheENversion)(DoesnotconcerntheFRversion)(DoesnotconcerntheITversion)(DoesnotconcerntheNLversion)(DoesnotconcernthePTversion)(DoesnotconcerntheSVversion)

Inpoint6.2(Safetyphrasesforsubstancesandpreparations):DE:

S28BeiBerührungmitderHautsofortmitviel󰂅abwaschen(vomHerstelleranzugeben)󰂗Anwendungsbereich:

󰂗sehrgiftige,giftigeoderätzendeStoffeundZubereitungen;󰂗Verwendung:

󰂗obligatorischfürsehrgiftigeStoffeundZubereitungen;

󰂗empfohlenfürsonstigeobengenannteStoffeundZubereitungen,inbesondere,wennWassernichtdiegeeignete

Spülflüssigkeitist;󰂗empfohlenfürätzendeStoffeundZubereitungen,dieandieallgemeineÖffentlichkeitabgegebenwerden.(DoesnotconcerntheESversion)(DoesnotconcerntheDAversion)(DoesnotconcerntheELversion)(DoesnotconcerntheENversion)(DoesnotconcerntheFRversion)(DoesnotconcerntheITversion)(DoesnotconcerntheNLversion)(DoesnotconcernthePTversion)(DoesnotconcerntheFIversion)(DoesnotconcerntheSVversion)

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FI:

ENOfficialJournaloftheEuropeanCommunities8.6.2000

S29Eisaatyhjentääviemäriin󰂗Soveltamisala:

󰂗erittäinhelpostisyttyvättaihelpostisyttyvätveteensekoittumattomatnesteet,󰂗erittäinmyrkyllisettaimyrkyllisetaineetjavalmisteet,󰂗ympäristöllevaarallisetaineet.󰂗Käytönperusteet:

󰂗pakollinenyleisessäkulutuksessatodennäkoisestikäytettävilleympäristöllevaarallisillejatunnuksellaNluokitel-luilleaineille,jolleikyseessäoleaineentarkoitettukäyttö,󰂗suositeltavayleisessäkulutuksessatodennäkoisestikäytettävillemuilleedellämainituilleaineilletaivalmisteille,

jolleikyseessäolekemikaalintarkoitettukäyttö.(DoesnotconcerntheESversion)(DoesnotconcerntheDAversion)(DoesnotconcerntheDEversion)(DoesnotconcerntheELversion)(DoesnotconcerntheENversion)(DoesnotconcerntheFRversion)(DoesnotconcerntheITversion)(DoesnotconcerntheNLversion)(DoesnotconcernthePTversion)(DoesnotconcerntheSVversion)

8.6.2000ENOfficialJournaloftheEuropeanCommunitiesL136/

ANNEX6󰂑ANNEXIXPARTA

Provisionsrelatingtochild-prooffastenings

InadditiontotheprovisionsinArticle22(1)(e)ofthisDirective,containersofwhatevercapacitycontainingsubstancespresentinganaspirationhazard(Xn;R65)andclassifiedandlabelledaccordingtoparagraph3.2.3ofAnnexVItothisDirective,withtheexceptionofsubstancesplacedonthemarketintheformofaerosolsorinacontainerfittedwithasealedsprayattachment,shallbefittedwithchild-prooffastenings.1.Reclosablepackages

Child-prooffasteningsusedonreclosablepackagesshallcomplywithISOstandard8317(1July19edition)relatingto󰂓Child-resistantpackages󰂗Requirementsandmethodsoftestingforreclosablepackages󰂔adoptedbytheInternationalStandardOrganisation(ISO).2.Non-reclosablepackages

Child-prooffasteningsusedonnon-reclosablepackagesshallcomplywithCENstandardEN862(March1997edition)relatingto󰂓Packaging󰂗Child-resistantpackaging󰂗Requirementsandtestingproceduresfornon-reclosablepackagesfornon-pharmaceuticalproducts󰂔adoptedbytheEuropeanCommitteeforStandardisation(CEN).3.Notes

1.2.

EvidenceofconformitywiththeabovestandardsmaybecertifiedonlybylaboratorieswhichconformwithEuropeanStandardsSeriesEN45000.Specificcases

Ifitseemsobviousthatpackagingissufficientlysafeforchildrenbecausetheycannotgetaccesstothecontentswithoutthehelpofatool,thetestdoesnotneedtobeperformed.

Inallothercasesandwhentherearesufficientgroundsfordoubtingthesecurityoftheclosureforachild,thenationalauthoritymayaskthepersonresponsibleforputtingtheproductonthemarkettogiveitacertificatefromalaboratory,describedin3.1,statingthateither:

󰂗thetypeofclosureissuchthatitisnotnecessarytotesttotheISOandCENstandardsreferredtoabove,

or

󰂗theclosurehasbeentestedandhasbeenfoundtoconformwiththestandardsreferredtoabove.

PARTB

Provisionsrelatingtotactilewarningdevices

ThetechnicalspecificationsfortactilewarningdevicesshallconformwithENISOstandard11683(1997edition)relatingto󰂓Packaging󰂗Tactilewarningsofdanger󰂗Requirements󰂔.󰂒